Substituted benzosulphonamides

ABSTRACT

The present invention relates to substituted benzosulphonamide compounds of general formula (I): in which R1, R2, R3, R4, R5 and A are as defined in the claims, to methods of preparing said compounds, to pharmaceutical compositions and combinations comprising said compounds and to the use of said compounds for manufacturing a pharmaceutical composition for the treatment or prophylaxis of a disease, in particular of a hyper-proliferative and/or angiogenesis disorder, as a sole agent or in combination with other active ingredients.

FIELD OF THE INVENTION

The present invention relates to substituted benzosulphonamides,(hereinafter referred to as “compounds of general formula (I)”) asdescribed and defined herein, to methods of preparing said compounds, topharmaceutical compositions and combinations comprising said compoundsand to the use of said compounds for manufacturing a pharmaceuticalcomposition for the treatment or prophylaxis of a disease, in particularof a hyper-proliferative and/or angiogenesis disorder, as a sole agentor in combination with other active ingredients.

BACKGROUND OF THE INVENTION

Cancer is a disease resulting from an abnormal growth of tissue. Certaincancers have the potential to invade into local tissues and alsometastasize to distant organs. This disease can develop in a widevariety of different organs, tissues, and cell types. Therefore, theterm “cancer” refers to a collection of over a thousand differentdiseases.

Over 4.4 million people worldwide were diagnosed with breast, colon,ovarian, lung, or prostate cancer in 2002 and over 2.5 million peopledied of these devastating diseases (Globocan 2002 Report). In the UnitedStates alone, over 1.25 million new cases and over 500,000 deaths fromcancer were predicted in 2005. The majority of these new cases wereexpected to be cancers of the colon (˜100,000), lung (˜170,000), breast(˜210,000) and prostate (˜230,000). Both the incidence and prevalence ofcancer is predicted to increase by approximately 15% over the next tenyears, reflecting an average growth rate of 1.4% [1].

Accumulating evidence suggests that cancer can be envisioned as a“signaling disease”, in which alterations in the cellular genomeaffecting the expression and/or function of oncogenes and tumorsuppressor genes would ultimately affect the transmission of signalsthat normally regulate cell growth, differentiation, and programmed celldeath (apoptosis). Unraveling the signaling pathways that aredysregulated in human cancers has resulted in the design of anincreasing number of mechanism-based therapeutic agents [2]. Signaltransduction inhibition as a therapeutic strategy for human malignancieshas recently met with remarkable success, as exemplified by thedevelopment of Gleevec for the treatment of chronic myelogenous leukemia(CML) and gastrointestinal stromal tumors (GIST), heralding a new era of“molecularly-targeted” therapies [3-5].

The mitogen-activated protein kinase (MAPK) module is a key integrationpoint along the signal transduction cascade that links diverseextracellular stimuli to proliferation, differentiation and survival.Scientific studies over the last twenty years have led to a quitedetailed molecular dissection of this pathway, which has now grown toinclude five different MAPK subfamilies [extracellular signal-regulatedkinases ERK-1/2, c-Jun-N-terminal kinases (JNKs), p38 kinases, ERK-3/4,and ERK-5], with distinct molecular and functional features [6-8]. Whilecertain subfamilies, such as the p38 family, are becoming therapeutictargets in inflammatory and degenerative diseases, the MAPK cascade thatproceeds from Ras to ERK-1/2 (the main mitogenic pathway initiated bypeptide growth factors) is starting to emerge as a prime target for themolecular therapy of different types of human cancers [9-11], The MAPKpathway is aberrantly activated in many human tumors as a result ofgenetic and epigenetic changes, resulting in increased proliferation andresistance to apoptotic stimuli. In particular, mutated oncogenic formsof Ras are found in 50% of colon and >90% of pancreatic cancers [12].Recently, BRAF mutations have been found in >60% of malignant melanoma[13]. These mutations result in a constitutively activated MAPK pathway.In addition, overexpression of or mutational activation of certainreceptor tyrosine kinases can also lead to increased activation of theRaf-MEK-ERK pathway.

The modular nature of the Raf/MEK/ERK cascade becomes less pleiotropicat the crossover point that is regulated by MEK [14]. No substrates forMEK have been identified other than ERK-1/2. Phosphorylated ERK is theproduct of MEK activity and thus its detection in cancer cells and intumor tissues provides a direct measure of MEK inhibition. Theselectivity of MEK for ERK1/2 coupled with the availability ofantibodies specific for the dually phosphorylated and activated form ofERK, makes MEK an attractive target for anticancer drug development. Inaddition, it was recently shown that MEK activation regulates matrixmineralization (Blood 2007, 40, 68), thereby modulation of MEK activitymay also be applicable for the treatment of diseases caused by oraccompanied with dysregulation of tissue mineralization, morespecifically for the treatment of diseases caused by or accompanied withdysregulation of bone mineralization.

First-generation MEK inhibitors, PD98059 [15] and U0126 [16], do notappear to compete with ATP and thus are likely to have distinct bindingsites on MEK; these is compounds have been extensively used in modelsystems in vitro and in vivo to attribute biological activities toERK1/2. A second-generation MEK1/2 inhibitor, PD184352 (now calledCI-1040), has an IC₅₀ in the low nanomolar range, enhancedbioavailability, and also appears to work via an allosteric, nonATP-competitive mechanism [17]. Oral treatment with CI-1040 has beenshown to inhibit colon cancer growth in vivo in mouse models [18] andthis compound was evaluated in phase I/II clinical trials in humanswhere it eventually failed because of insufficient efficacy [19].Further allosteric MEK inhibitors have recently entered the clinic butwere found to have limitations such as poor exposure profiles, limitedefficacy and/or toxicity issues. Small molecules MEK inhibitors havebeen disclosed, including in U.S. Patent Publications Nos. 2003/0232869,2004/0116710, 2003/0216420 and in U.S. patent application Ser. Nos.10/654,580 and 10/929, 295 each of which is hereby incorporated byreference. A number of additional patent applications have appeared inthe last few years including U.S. Pat. No. 5,525,6625; WO 98/43960; WO99/01421; WO 99/01426; WO 00/41505; WO 00/41994; WO 00/42002; WO00/42003; WO 00/42022; WO 00/42029; WO 00/68201; WO 01/68619; WO02/06213; WO 03/077914; WO 03/077855; WO 04/083167; WO 05/0281126; WO05/051301; WO 05/121142; WO 06/114466; WO 98/37881; WO 00/35435; WO00/35436; WO 00/40235; WO 00/40237; WO 01/05390; WO 01/05391; WO01/05392; WO 01/05393; WO 03/062189; WO 03/062191; WO 04/056789; WO05/000818; WO 05/007616; WO 05/009975; WO 05/051300; WO05/051302; WO05/028426; WO 06/056427; WO 03/035626; and WO 06/029862.

Despite advancements in the art, there remains a need for cancertreatments and anti-cancer compounds. More specifically, there remains aneed for structurally novel MEK inhibitors with a balancedpotency-properties profile. It would be especially desirable to identifynovel MEK inhibitors which incorporate structural motifs which have notbeen previously exemplified as being compatible with potent MEKinhibition. It would be especially favorable if these structural motifswould further allow for is improvement of MEK potency and/or modulationof compound properties (including physico-chemical, pharmacodynamicaland pharmacokinetical properties).

WO 2008/138639 (Bayer Schering Pharma Aktiengesellschaft) relates tosubstituted phenylaminobenzene compounds, pharmaceutical compositionscontaining such compounds and the use of such compounds or compositionsfor treating hyperproliferative and/or angiogenesis disorders. Saidcompounds were found to be potent and selective MEK inhibitors. Saidcompounds are derived from a 1-substituted-2-phenylamino-phenyl scaffoldwith a further specifically substituted side chain in the 6-position ofthe phenyl scaffold. This finding was surprising as inspection ofpublished phenyl-scaffold-derived MEK inhibitors and previousstructure-activity relationship analysis (see for example HaileTecle/Pfizer Global Research: “MEK inhibitors”, presented at DrewUniversity, 15 Jun. 2006) suggested that in phenyl-scaffold-based MEKinhibitors larger 6-substituents are detrimental for achieving high MEKinhibitory potency. Said compounds are potent MEK inhibitors and inhibitactivation of the MEK-ERK pathway.

However, none of the state of the art described above describes theselected compounds of general formula (I) of the present invention,which bear a selected substituent in 1-position of the central phenylring (a selected sulphonamide group of general formula —NHS(O)₂R3), andbear a selected substituent in the 3-position of the western phenyl ring(a specially selected sulphonamide group of general formula —NHS(O)₂R4),or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or asalt thereof, or a mixture of same, as described and defined herein, andas hereinafter referred to as “compounds of the present invention”, ortheir pharmacological activity.

It has now been found, and this constitutes the basis of the presentinvention, that said compounds of the present invention have surprisingand advantageous properties.

In particular, said compounds of the present invention have surprisinglybeen found to effectively strongly inhibit cancer cell proliferation notonly in B-Raf mutated human A375 melanoma cells, but also inK-Ras-mutated human A549 non small cell lung cancer cells and in K-Rasmutated human HCT116 colorectal cancer cells. More surprisingly,compounds of the present invention exhibit a stronger cancer cellproliferation inhibition in comparison to that of a compound from priorart document WO 2008/138639.

In view of this, said compounds of general formula (I) of the presentinvention may therefore be used for the treatment or prophylaxis ofdiseases of uncontrolled cell growth, proliferation and/or survival,inappropriate cellular immune responses, or inappropriate cellularinflammatory responses or diseases which are accompanied withuncontrolled cell growth, proliferation and/or survival, inappropriatecellular immune responses, or inappropriate cellular inflammatoryresponses, particularly in which the uncontrolled cell growth,proliferation and/or survival, inappropriate cellular immune responses,or inappropriate cellular inflammatory responses is mediated by themitogen activated protein kinase (MEK-ERK) pathway, such as, forexample, haemotological tumours, solid tumours, and/or metastasesthereof, e.g. leukaemias and myelodysplastic syndrome, malignantlymphomas, head and neck tumours including brain tumours and brainmetastases, tumours of the thorax including non-small cell and smallcell lung tumours, gastrointestinal tumours, endocrine tumours, mammaryand other gynaecological tumours, urological tumours including renal,bladder and prostate tumours, skin tumours, and sarcomas, and/ormetastases thereof.

DESCRIPTION OF THE INVENTION

In accordance with a first aspect, the present invention coverscompounds of general formula (I):

in which:

-   R1 is a hydrogen atom or a fluorine atom;-   R2 is a halogen atom or a C₂-alkynyl group;-   R3 is an —NH₂, —NH(C₁-C₆-alkyl), —N(C₁-C₆-alkyl)₂, —C₁-C₆-alkyl, or    —C₃-C₆-cycloalkyl group;-   R4 is an —NH₂, —NH(C₁-C₆-alkyl), —N(C₁-C₆-alkyl)₂, —C₁-C₆-alkyl, or    —C₃-C₆-cycloalkyl group;-   R5 is a halogen atom, or a —C₁-C₆-alkyl or —O—C₁-C₆-alkyl group;-   A is —(CH₂)_(n)—, in which n=0, or 1;    or a tautomer, stereoisomer, N-oxide, salt, hydrate, solvate,    metabolite, or prodrug thereof.

DEFINITIONS

The terms as mentioned in the present text have preferably the followingmeanings:

The term “halogen atom” or “halo” is to be understood as meaning afluorine, chlorine, bromine or iodine atom.

The term “C₁-C₁₀-alkyl” is to be understood as preferably meaning alinear or branched, saturated, monovalent hydrocarbon group having 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, particularly 1, 2, 3, 4, 5 or 6carbon atoms, e.g. a methyl, ethyl, propyl, butyl, pentyl, hexyl,iso-propyl, iso-butyl, sec-butyl, tert-butyl, iso-pentyl, 2-methylbutyl,1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neo-pentyl,1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl,1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl,2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl,1,3-dimethylbutyl, or 1,2-dimethylbutyl group, or an isomer thereof.Particularly, said group has 1, 2 or 3 carbon atoms (“C₁-C₃-alkyl”),methyl, ethyl, n-propyl- or iso-propyl.

The term “halo-C₁-C₁₀-alkyl” is to be understood as preferably meaning alinear or branched, saturated, monovalent hydrocarbon group in which theterm “C₁-C₁₀-alkyl” is defined supra, and in which one or more hydrogenatoms is replaced by a halogen atom, in identically or differently, i.e.one halogen atom being independent from another. Particularly, saidhalogen atom is F. Said halo-C₁-C₁₀-alkyl group is, for example, —CF₃,—CHF₂, —CH₂F, —CF₂CF₃, or —CH₂CF₃.

The term “C₁-C₁₀-alkoxy” is to be understood as preferably meaning alinear or branched, saturated, monovalent, hydrocarbon group of formula—O-alkyl, in which the term “alkyl” is defined supra, e.g. a methoxy,ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, tert-butoxy,sec-butoxy, pentoxy, iso-pentoxy, or n-hexoxy group, or an isomerthereof.

The term “halo-C₁-C₁₀-alkoxy” is to be understood as preferably meaninga linear or branched, saturated, monovalent C₁-C₁₀-alkoxy group, asdefined supra, in which one or more of the hydrogen atoms is replaced,in identically or differently, by a halogen atom. Particularly, saidhalogen atom is F. Said halo-C₁-C₁₀alkoxy group is, for example, —OCF₃,—OCHF₂, —OCH₂F, —OCF₂CF₃, or —OCH₂CF₃.

The term “C₁-C₁₀-alkoxy-C₁-C₁₀-alkyl” is to be understood as preferablymeaning a linear or branched, saturated, monovalent alkyl group, asdefined supra, in which one or more of the hydrogen atoms is replaced,in identically or differently, by a C₁-C₁₀-alkoxy group, as definedsupra, e.g. methoxyalkyl, ethoxyalkyl, propyloxyalkyl, iso-propoxyalkyl,butoxyalkyl, iso-butoxyalkyl, tert-butoxyalkyl, sec-butoxyalkyl,pentyloxyalkyl, iso-pentyloxyalkyl, hexyloxyalkyl group, in which theterm “C₁-C_(1r) alkyl” is defined supra, or an isomer thereof.

The term “halo-C₁-C₁₀-alkoxy-C₁-C₁₀-alkyl” is to be understood aspreferably meaning a linear or branched, saturated, monovalentC₁-C₁₀-alkoxy-C₁-C₁₀-alkyl group, as defined supra, in which one or moreof the hydrogen atoms is replaced, in identically or differently, by ahalogen atom. Particularly, said halogen atom is F. Saidhalo-C₁-C₁₀-alkoxy-C₁-C₁₀-alkyl group is, for example, —CH₂CH₂OCF₃,—CH₂CH₂OCHF₂, —CH₂CH₂OCH₂F, —CH₂CH₂OCF₂CF₃, or —CH₂CH₂OCH₂CF₃.

The term “C₂-C₁₀-alkenyl” is to be understood as preferably meaning alinear or branched, monovalent hydrocarbon group, which contains one ormore double bonds, and which has 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbonatoms, particularly 2 or 3 carbon atoms (“C₂-C₃-alkenyl”), it beingunderstood that in the case in which said alkenyl group contains morethan one double bond, then said double bonds may be isolated from, orconjugated with, each other. Said alkenyl group is, for example, avinyl, allyl, (E)-2-methylvinyl, (Z)-2-methylvinyl, homoallyl,(E)-but-2-enyl, (Z)-but-2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl,pent-4-enyl, (E)-pent-3-enyl, (Z)-pent-3-enyl, (E)-pent-2-enyl,(Z)-pent-2-enyl, (E)-pent-1-enyl, (Z)-pent-1-enyl, hex-5-enyl,(E)-hex-4-enyl, (Z)-hex-4-enyl, (E)-hex-3-enyl, (Z)-hex-3-enyl,(E)-hex-2-enyl, (Z)-hex-2-enyl, (E)-hex-1-enyl, (Z)-hex-1-enyl,isopropenyl, 2-methylprop-2-enyl, 1-methylprop-2-enyl,2-methylprop-1-enyl, (E)-1-methylprop-1-enyl, (Z)-1-methylprop-1-enyl,3-methylbut-3-enyl, 2-methylbut-3-enyl, 1-methylbut-3-enyl,3-methylbut-2-enyl, (E)-2-methylbut-2-enyl, (Z)-2-methylbut-2-enyl,(E)-1-methylbut-2-enyl, (Z)-1-methylbut-2-enyl, (E)-3-methylbut-1-enyl,(Z)-3-methylbut-1-enyl, (E)-2-methylbut-1-enyl, (Z)-2-methylbut-1-enyl,(E)-1-methylbut-1-enyl, (Z)-1-methylbut-1-enyl, 1,1-dimethylprop-2-enyl,1-ethylprop-1-enyl, 1-propylvinyl, 1-isopropylvinyl,4-methylpent-4-enyl, 3-methylpent-4-enyl, 2-methylpent-4-enyl,1-methylpent-4-enyl, 4-methylpent-3-enyl, (E)-3-methylpent-3-enyl,(Z)-3-methylpent-3-enyl, (E)-2-methylpent-3-enyl,(Z)-2-methylpent-3-enyl, (E)-1-methylpent-3-enyl,(Z)-1-methylpent-3-enyl, (E)-4-methylpent-2-enyl,(Z)-4-methylpent-2-enyl, (E)-3-methylpent-2-enyl,(Z)-3-methylpent-2-enyl, (E)-2-methylpent-2-enyl,(Z)-2-methylpent-2-enyl, (E)-1-methylpent-2-enyl,(Z)-1-methylpent-2-enyl, (E)-4-methylpent-1-enyl,(Z)-4-methylpent-1-enyl, (E)-3-methylpent-1-enyl,(Z)-3-methylpent-1-enyl, (E)-2-methylpent-1-enyl,(Z)-2-methylpent-1-enyl, (E)-1-methylpent-1-enyl,(Z)-1-methylpent-1-enyl, 3-ethylbut-3-enyl, 2-ethylbut-3-enyl,1-ethylbut-3-enyl, (E)-3-ethylbut-2-enyl, (Z)-3-ethylbut-2-enyl,(E)-2-ethylbut-2-enyl, (Z)-2-ethylbut-2-enyl, (E)-1-ethylbut-2-enyl,(Z)-1-ethylbut-2-enyl, (E)-3-ethylbut-1-enyl, (Z)-3-ethylbut-1-enyl,2-ethylbut-1-enyl, (E)-1-ethylbut-1-enyl, (Z)-1-ethylbut-1-enyl,2-propylprop-2-enyl, 1-propylprop-2-enyl, 2-isopropylprop-2-enyl,1-isopropylprop-2-enyl, (E)-2-propylprop-1-enyl,(Z)-2-propylprop-1-enyl, (E)-1-propylprop-1-enyl,(Z)-1-propylprop-1-enyl, (E)-2-isopropylprop-1-enyl,(Z)-2-isopropylprop-1-enyl, (E)-1-isopropylprop-1-enyl,(Z)-1-isopropylprop-1-enyl, (E)-3,3-dimethylprop-1-enyl,(Z)-3,3-dimethylprop-1-enyl, 1-(1,1-dimethylethyl)ethenyl,buta-1,3-dienyl, penta-1,4-dienyl, hexa-1,5-dienyl, or methylhexadienylgroup. Particularly, said group is vinyl or allyl.

The term “C₂-C₁₀-alkynyl” is to be understood as preferably meaning alinear or branched, monovalent hydrocarbon group which contains one ormore triple bonds, and which contains 2, 3, 4, 5, 6, 7, 8, 9 or 10carbon atoms, particularly 2 or 3 carbon atoms (“C₂-C₃-alkynyl”). SaidC₂-C₁₀-alkynyl group is, for example, ethynyl, prop-1-ynyl, prop-2-ynyl,but-1-ynyl, but-2-ynyl, but-3-ynyl, pent-1-ynyl, pent-2-ynyl,pent-3-ynyl, pent-4-ynyl, hex-1-ynyl, hex-2-inyl, hex-3-inyl,hex-4-ynyl, hex-5-ynyl, 1-methylprop-2-ynyl, 2-methylbut-3-ynyl,1-methylbut-3-ynyl, 1-methylbut-2-ynyl, 3-methylbut-1-ynyl,1-ethylprop-2-ynyl, 3-methylpent-4-ynyl, 2-methylpent-4-ynyl,1-methylpent-4-ynyl, 2-methylpent-3-ynyl, 1-methylpent-3-ynyl,4-methylpent-2-ynyl, 1-methylpent-2-ynyl, 4-methylpent-1-ynyl,3-methylpent-1-ynyl, 2-ethylbut-3-ynyl, 1-ethylbut-3-ynyl,1-ethylbut-2-ynyl, 1-propylprop-2-ynyl, 1-isopropylprop-2-ynyl,2,2-dimethylbut-3-inyl, 1,1-dimethylbut-3-ynyl, 1,1-dimethylbut-2-ynyl,or 3,3-dimethyl-but-1-ynyl group. Particularly, said alkynyl group isethynyl, prop-1-ynyl, or prop-2-inyl.

The term “C₃-C₁₀-cycloalkyl” is to be understood as preferably meaning asaturated, monovalent, mono-, or bicyclic hydrocarbon ring whichcontains 3, 4, 5, 6, 7, 8, 9, or carbon atoms, particularly 3, 4, 5, or6 carbon atoms (“C₃-C₆-cycloalkyl”). Said C₃-C₁₀-cycloalkyl group is forexample, a monocyclic hydrocarbon ring, e.g. a cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl orcyclodecyl group, or a bicyclic hydrocarbon ring, e.g. aperhydropentalenylene or decalin ring. Said cycloalkyl ring canoptionally contain one or more double bonds e.g. cycloalkenyl, such as acyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,cyclooctenyl, cyclononenyl, or cyclodecenyl group, wherein the bondbetween said ring with the rest of the molecule may be to any carbonatom of said ring, be it saturated or unsaturated.

The term “alkylene” is understood as preferably meaning an optionallysubstituted hydrocarbon chain (or “tether”) having 1, 2, 3, 4, 5, or 6carbon atoms, i.e. an optionally substituted —CH₂— (“methylene” or“single membered tether” or, for example —C(Me)₂-), —CH₂—CH₂—(“ethylene”, “dimethylene”, or “two-membered tether”), —CH₂—CH₂—CH₂—(“propylene”, “trimethylene”, or “three-membered tether”),—CH₂—CH₂—CH₂—CH₂— (“butylene”, “tetramethylene”, or “four-memberedtether”), —CH₂—CH₂—CH₂—CH₂—CH₂— (“pentylene”, “pentamethylene” or“five-membered ether”), or —CH₂—CH₂—CH₂—CH₂—CH₂—CH₂— (“hexylene”,“hexamethylene”, or six-membered tether”) group. Particularly, saidalkylene tether has 1, 2, 3, 4, or 5 carbon atoms, more particularly 1or 2 carbon atoms.

The term “C₁-C₆”, as used throughout this text, e.g. in the context ofthe definition of “C₁-C₆-alkyl”, “C₁-C₆-haloalkyl”, “C₁-C₆-alkoxy”, or“C₁-C₆-haloalkoxy” is to be understood as meaning an alkyl group havinga finite number of carbon atoms of 1 to 6, i.e. 1, 2, 3, 4, 5, or 6carbon atoms. It is to be understood further that said term “C₁-C₆” isto be interpreted as any sub-range comprised therein, e.g. C₁-C₆, C₂-C₅;C₃-C₄, C₁-C₂, C₁-C₃, C₁-C₄, C₁-C₅, C₁-C₆; particularly C₁-C₂, C₁-C₃,C₁-C₄, C₁-C₅, C₁-C₆; more particularly C₁-C₄, in the case of“C₁-C₆-haloalkyl” or “C₁-C₆-haloalkoxy” even more particularly C₁-C₂.

Similarly, as used herein, the term “C₂-C₆”, as used throughout thistext, e.g. in the context of the definitions of “C₂-C₆-alkenyl” and“C₂-C₆-alkynyl”, is to be understood as meaning an alkenyl group or analkynyl group having a finite number of carbon atoms of 2 to 6, i.e. 2,3, 4, 5, or 6 carbon atoms. It is to be understood further that saidterm “C₂-C₆” is to be interpreted as any sub-range comprised therein,e.g. C₂-C₆, C₃-C₅, C₃-C₄, C₂-C₃, C₂-C₄, C₂-C₅; particularly C₂-C₃.

Further, as used herein, the term “C₃-C₁₀”, as used throughout thistext, e.g. in the context of the definition of “C₃-C₁₀-cycloalkyl”, isto be understood as meaning a cycloalkyl group having a finite number ofcarbon atoms of 3 to 10, i.e. 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms,particularly 3, 4, 5 or 6 carbon atoms. It is to be understood furtherthat said term “C₃-C₁₀” is to be interpreted as any sub-range comprisedtherein, e.g. C₃-C₁₀, C₄-C₉, C₅-C₈, C₆-C₇; particularly C₃-C₆.

As used herein, the term “one or more times”, e.g. in the definition ofthe substituents of the compounds of the general formulae of the presentinvention, is understood as meaning “one, two, three, four or fivetimes, particularly one, two, three or four times, more particularlyone, two or three times, even more particularly one or two times”.

Where the plural form of the word compounds, salts, polymorphs,hydrates, solvates and the like, is used herein, this is taken to meanalso a single compound, salt, polymorph, isomer, hydrate, solvate or thelike.

The compounds of this invention may contain one or more asymmetriccentre, depending upon the location and nature of the varioussubstituents desired. Asymmetric carbon atoms may be present in the (R)or (S) configuration, resulting in racemic mixtures in the case of asingle asymmetric centre, and diastereomeric mixtures in the case ofmultiple asymmetric centres. In certain instances, asymmetry may also bepresent due to restricted rotation about a given bond, for example, thecentral bond adjoining two substituted aromatic rings of the specifiedcompounds.

Substituents on a ring may also be present in either cis or trans form.It is intended that all such configurations (including enantiomers anddiastereomers), are included within the scope of the present invention.

Preferred compounds are those which produce the more desirablebiological activity. Separated, pure or partially purified isomers andstereoisomers or racemic or diastereomeric mixtures of the compounds ofthis invention are also included within the scope of the presentinvention. The purification and the separation of such materials can beaccomplished by standard techniques known in the art.

The optical isomers can be obtained by resolution of the racemicmixtures according to conventional processes, for example, by theformation of diastereoisomeric salts using an optically active acid orbase or formation of covalent diastereomers. Examples of appropriateacids are tartaric, diacetyltartaric, ditoluoyltartaric andcamphorsulfonic acid. Mixtures of diastereoisomers can be separated intotheir individual diastereomers on the basis of their physical and/orchemical differences by methods known in the art, for example, bychromatography or fractional crystallisation. The optically active basesor acids are then liberated from the separated diastereomeric salts. Adifferent process for separation of optical isomers involves the use ofchiral chromatography (e.g., chiral HPLC columns), with or withoutconventional derivatisation, optimally chosen to maximise the separationof the enantiomers. Suitable chiral HPLC columns are manufactured byDiacel, e.g., Chiracel OD and Chiracel OJ among many others, allroutinely selectable. Enzymatic separations, with or withoutderivatisation, are also useful. The optically active compounds of thisinvention can likewise be obtained by chiral syntheses utilizingoptically active starting materials.

In order to limit different types of isomers from each other referenceis made to IUPAC Rules Section E (Pure Appl Chem 45, 11-30, 1976).

The present invention includes all possible stereoisomers of thecompounds of the present invention as single stereoisomers, or as anymixture of said stereoisomers, in any ratio. Isolation of a singlestereoisomer, e.g. a single enantiomer or a single diastereomer, of acompound of the present invention may be achieved by any suitable stateof the art method, such as chromatography, especially chiralchromatography, for example.

Further, the compounds of the present invention may exist as tautomers.For example, any compound of the present invention which contains apyrazole moiety as a heteroaryl group for example can exist as a 1Htautomer, or a 2H tautomer, or even a mixture in any amount of the twotautomers, or a triazole moiety for example can exist as a 1H tautomer,a 2H tautomer, or a 4H tautomer, or even a mixture in any amount of said1H, 2H and 4H tautomers, viz.:

The present invention includes all possible tautomers of the compoundsof the present invention as single tautomers, or as any mixture of saidtautomers, in any ratio.

Further, the compounds of the present invention can exist as N-oxides,which are defined in that at least one nitrogen of the compounds of thepresent invention is oxidised. The present invention includes all suchpossible N-oxides.

The present invention also relates to useful forms of the compounds asdisclosed herein, such as metabolites, hydrates, solvates, prodrugs,salts, in particular pharmaceutically acceptable salts, andco-precipitates.

The compounds of the present invention can exist as a hydrate, or as asolvate, wherein the compounds of the present invention contain polarsolvents, in particular water, methanol or ethanol for example asstructural element of the crystal lattice of the compounds. The amountof polar solvents, in particular water, may exist in a stoichiometric ornon-stoichiometric ratio. In the case of stoichiometric solvates, e.g. ahydrate, hemi-, (semi-), mono-, sesqui-, di-, tri-, tetra-, penta- etc.solvates or hydrates, respectively, are possible. The present inventionincludes all such hydrates or solvates.

Further, the compounds of the present invention can exist in free form,e.g. as a free base, or as a free acid, or as a zwitterion, or can existin the form of a salt. Said salt may be any salt, either an organic orinorganic addition salt, particularly any pharmaceutically acceptableorganic or inorganic addition salt, customarily used in pharmacy.

The term “pharmaceutically acceptable salt” refers to a relativelynon-toxic, inorganic or organic acid addition salt of a compound of thepresent invention. For example, see S. M. Berge, et al. “PharmaceuticalSalts,” J. Pharm. Sci. 1977, 66, 1-19.

A suitable pharmaceutically acceptable salt of the compounds of thepresent invention may be, for example, an acid-addition salt of acompound of the present invention bearing a nitrogen atom, in a chain orin a ring, for example, which is sufficiently basic, such as anacid-addition salt with an inorganic acid, such as hydrochloric,hydrobromic, hydroiodic, sulfuric, bisulfuric, phosphoric, or nitricacid, for example, or with an organic acid, such as formic, acetic,acetoacetic, pyruvic, trifluoroacetic, propionic, butyric, hexanoic,heptanoic, undecanoic, lauric, benzoic, salicylic,2-(4-hydroxybenzoyl)-benzoic, camphoric, cinnamic,cyclopentanepropionic, digluconic, 3-hydroxy-2-naphthoic, nicotinic,pamoic, pectinic, persulfuric, 3-phenylpropionic, picric, pivalic,2-hydroxyethanesulfonate, itaconic, sulfamic, trifluoromethanesulfonic,dodecylsulfuric, ethansulfonic, benzenesulfonic, para-toluenesulfonic,methansulfonic, 2-naphthalenesulfonic, naphthalinedisulfonic,camphorsulfonic acid, citric, tartaric, stearic, lactic, oxalic,malonic, succinic, malic, adipic, alginic, maleic, fumaric, D-gluconic,mandelic, ascorbic, glucoheptanoic, glycerophosphoric, aspartic,sulfosalicylic, hemisulfuric, or thiocyanic acid, for example.

Further, another suitably pharmaceutically acceptable salt of a compoundof the present invention which is sufficiently acidic, is an alkalimetal salt, for example a sodium or potassium salt, an alkaline earthmetal salt, for example a calcium or magnesium salt, an ammonium salt ora salt with an organic base which affords a physiologically acceptablecation, for example a salt with N-methyl-glucamine, dimethyl-glucamine,ethyl-glucamine, lysine, dicyclohexylamine, 1,6-hexadiamine,ethanolamine, glucosamine, sarcosine, serinol,tris-hydroxy-methyl-aminomethane, aminopropandiol, sovak-base,1-amino-2,3,4-butantriol. Additionally, basic nitrogen containing groupsmay be quaternised with such agents as lower alkyl halides such asmethyl, ethyl, propyl, and butyl chlorides, bromides and iodides;dialkyl sulfates like dimethyl, diethyl, and dibutyl sulfate; and diamylsulfates, long chain halides such as decyl, lauryl, myristyl andstrearyl chlorides, bromides and iodides, aralkyl halides like benzyland phenethyl bromides and others.

Those skilled in the art will further recognise that acid addition saltsof the claimed compounds may be prepared by reaction of the compoundswith the appropriate inorganic or organic acid via any of a number ofknown methods. Alternatively, alkali and alkaline earth metal salts ofacidic compounds of the invention are prepared by reacting the compoundsof the invention with the appropriate base via a variety of knownmethods.

The present invention includes all possible salts of the compounds ofthe present invention as single salts, or as any mixture of said salts,in any ratio.

As used herein, the term “in vivo hydrolysable ester” is understood asmeaning an in vivo hydrolysable ester of a compound of the presentinvention containing a carboxy or hydroxy group, for example, apharmaceutically acceptable ester which is hydrolysed in the human oranimal body to produce the parent acid or alcohol. Suitablepharmaceutically acceptable esters for carboxy include for examplealkyl, cycloalkyl and optionally substituted phenylalkyl, in particularbenzyl esters, C₁-C₆ alkoxymethyl esters, e.g. methoxymethyl, C₁-C₆alkanoyloxymethyl esters, e.g. pivaloyloxymethyl, phthalidyl esters,C₃-C₈ cycloalkoxy-carbonyloxy-C₁-C₆ alkyl esters, e.g.1-cyclohexylcarbonyloxyethyl; 1,3-dioxolen-2-onylmethyl esters, e.g.5-methyl-1,3-dioxolen-2-onylmethyl; and C₁-C₆-alkoxycarbonyloxyethylesters, e.g. 1-methoxycarbonyloxyethyl, and may be formed at any carboxygroup in the compounds of this invention.

An in vivo hydrolysable ester of a compound of the present inventioncontaining a hydroxy group includes inorganic esters such as phosphateesters and [alpha]-acyloxyalkyl ethers and related compounds which as aresult of the in vivo hydrolysis of the ester breakdown to give theparent hydroxy group. Examples of [alpha]-acyloxyalkyl ethers includeacetoxymethoxy and 2,2-dimethylpropionyloxymethoxy. A selection of invivo hydrolysable ester forming groups for hydroxy include alkanoyl,benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl,alkoxycarbonyl (to give alkyl carbonate esters), dialkylcarbamoyl andN-(dialkylaminoethyl)-N-alkylcarbamoyl (to give carbamates),dialkylaminoacetyl and carboxyacetyl. The present invention covers allsuch esters.

Furthermore, the present invention includes all possible crystallineforms, or polymorphs, of the compounds of the present invention, eitheras single polymorphs, or as a mixture of more than one polymorphs, inany ratio.

In accordance with a second aspect, the present invention coverscompounds of general formula (I), supra, in which:

-   R1 is a hydrogen atom or a fluorine atom;-   R2 is a fluorine atom or a C₂-alkynyl group;-   R3 is an —NH₂, —NH(C₁-C₆-alkyl), —N(C₁-C₆-alkyl)₂, —C₁-C₆-alkyl, or    —C₃-C₆-cycloalkyl group;-   R4 is an —NH₂, —NH(C₁-C₆-alkyl), —N(C₁-C₆-alkyl)₂, —C₁-C₆-alkyl, or    —C₃-C₆-cycloalkyl group;-   R5 is a fluorine atom or a methyl group;-   A is —(CH₂)_(n)—, in which n=0, or 1;    or a tautomer, stereoisomer, N-oxide, salt, hydrate, solvate,    metabolite, or prodrug thereof.

In accordance with a third aspect, the present invention coverscompounds of general formula (I), supra, in which:

-   R1 is a hydrogen atom or a fluorine atom;-   R2 is a fluorine atom or a C₂-alkynyl group;-   R3 is an —NH₂, —NH(C₁-C₆-alkyl), —N(C₁-C₆-alkyl)₂, —C₁-C₆-alkyl, or    —C₃-C₆-cycloalkyl group;-   R4 is an —NH₂, methyl, ethyl, n-propyl, iso-propyl or cyclopropyl or    cyclobutyl group;-   R5 is a fluorine atom or a methyl group;-   A is —(CH₂)_(n)—, in which n=0, or 1;    or a tautomer, stereoisomer, N-oxide, salt, hydrate, solvate,    metabolite, or prodrug thereof.

In accordance with a fourth aspect, the present invention coverscompounds of general formula (I), supra, in which:

-   R1 is a hydrogen atom or a fluorine atom;-   R2 is a fluorine atom or a C₂-alkynyl group;-   R3 is an —NH₂, methyl, ethyl, n-propyl, iso-propyl, cyclopropyl or    cyclobutyl group;-   R4 is an —NH₂, methyl, ethyl, n-propyl, iso-propyl, cyclopropyl or    cyclobutyl group;-   R5 is a fluorine atom or a methyl group;-   A is —(CH₂)_(n)—, in which n=0, or 1;    or a tautomer, stereoisomer, N-oxide, salt, hydrate, solvate,    metabolite, or prodrug thereof.

In a further embodiment of the above-mentioned aspects, the inventionrelates to compounds of formula (I), wherein R¹ is a hydrogen atom or afluorine atom.

In a further embodiment of the above-mentioned aspects, the inventionrelates to compounds of formula (I), wherein R2 is a halogen atom or aC₂-alkynyl group.

In a further embodiment of the above-mentioned aspects, the inventionrelates to compounds of formula (I), wherein R3 is an —NH₂,—NH(C₁-C₆-alkyl), —N(C₁-C₆-alkyl)₂, —C₁-C₆-alkyl, or —C₃-C₆-cycloalkylgroup.

In a further embodiment of the above-mentioned aspects, the inventionrelates to compounds of formula (I), wherein R4 is an —NH₂,—NH(C₁-C₆-alkyl), —N(C₁-C₆-alkyl)₂, —C₁-C₆-alkyl, or —C₃-C₆-cycloalkylgroup.

In a further embodiment of the above-mentioned aspects, the inventionrelates to compounds of formula (I), wherein R5 is a halogen atom, or a—C₁-C₆-alkyl or —O—C₁-C₆-alkyl group.

In a further embodiment of the above-mentioned aspects, the inventionrelates to compounds of formula (I), wherein A is —(CH₂)_(n)—, in whichn=0.

In a further embodiment of the above-mentioned aspects, the inventionrelates to compounds of formula (I), wherein A is —(CH₂)_(n)—, in whichn=1.

In a further embodiment of the above-mentioned aspects, the inventionrelates to compounds of formula (I), wherein R5 is a fluorine atom or amethyl group.

In a further embodiment of the above-mentioned aspects, the inventionrelates to compounds of formula (I), wherein R4 is an —NH₂, methyl,ethyl, n-propyl, iso-propyl, cyclopropyl or cyclobutyl group.

In a further embodiment of the above-mentioned aspects, the inventionrelates to compounds of formula (I), wherein R3 is an —NH₂, methyl,ethyl, n-propyl, iso-propyl, cyclopropyl or cyclobutyl group.

It is to be understood that the present invention relates to anysub-combination within any embodiment of the present invention ofcompounds of general formula (I), supra.

In a further aspect, the present invention covers compounds of generalformula (I) which are disclosed in the Example section of this text,infra.

In accordance with another aspect, the present invention covers a methodof preparing compounds of the present invention, the method comprisingthe steps as described herein.

In accordance with a further aspect, the present invention coversintermediate compounds which are useful in the preparation of compoundsof the present invention of general formula (I), particularly in themethod described herein. In particular, the present invention coverscompounds of general formula (4):

in which R1, R2, R4, R5 and A are as defined supra as for generalformula (I), and compounds of general formula (8):

in which R1, R2, R3, R5 and A are as defined supra as for generalformula (I), and compounds of general formula (12):

in which R1, R2, R3, R5 and A are as defined supra for general formula(I), and PG represents an acid-labile protecting group.

In accordance with a further aspect, the present invention relates tothe use of the intermediate compound of general formula (4), supra, orof the intermediate compound of general formula (8), supra, or of theintermediate compound of general formula (12), supra, for thepreparation of a compound of general formula (I), supra.

Experimental Details and General Processes Abbreviations and Acronyms

A comprehensive list of the abbreviations used by organic chemists ofordinary skill in the art appears in The ACS Style Guide (third edition)or the Guidelines for Authors for the Journal of Organic Chemistry. Theabbreviations contained in said lists, and all abbreviations utilized byorganic chemists of ordinary skill in the art are hereby incorporated byreference. For purposes of this invention, the chemical elements areidentified in accordance with the Periodic Table of the Elements, CASversion, Handbook of Chemistry and Physics, 67th Ed., 1986-87.

More specifically, when the following abbreviations are used throughoutthis disclosure, they have the following meanings:

-   Ac₂O acetic anhydride-   ACN acetonitrile-   AcO (or OAc) acetate-   anhyd anhydrous-   aq aqueous-   Ar aryl-   atm atmosphere-   ATP adenosine triphosphate-   b.i.d. twice a day-   Biotage silica gel chromatographic system, Biotage Inc.-   Bn benzyl-   bp boiling point-   Bz benzoyl-   BOC tert-butoxycarbonyl-   n-BuOH n-butanol-   t-BuOH tert-butanol-   t-BuOK potassium tert-butoxide-   calcd calculated-   CDI carbonyl diimidazole-   CD₃OD methanol-d₄-   Celite® diatomaceous earth filter agent, Celite Corp.-   CI-MS chemical ionization mass spectroscopy-   ¹³C NMR carbon-13 nuclear magnetic resonance-   conc concentrated-   DCC dicyclohexylcarbodiimide-   DCE dichloroethane-   DCM dichloromethane-   dec decomposition-   DIBAL diisobutylaluminum hydroxide-   DMAP 4-(N,N-dimethylamino)pyidine-   DME 1,2-dimethoxyethane-   DMF N,N-dimethylformamide-   DMSO dimethylsulfoxide-   DTT dithiothreitol-   E entgegen (configuration)-   e.g. for example-   EO electron impact-   ELSD evaporative light scattering detector-   eq equivalent-   ERK extracellular signal-regulated kinase-   ESI electrospray ionisation-   ES-MS electrospray mass spectroscopy-   et al. and others-   EtOAc ethyl acetate-   EtOH ethanol (100%)-   EtSH ethanethiol-   Et₂O diethyl ether-   Et₃N triethylamine-   GC gas chromatography-   GC-MS gas chromatography-mass spectroscopy-   h hour, hours-   ¹H NMR proton nuclear magnetic resonance-   HCl hydrochloric acid-   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-   Hex hexane-   HMPA hexamethylphosphoramide-   HMPT hexamethylphosphoric triamide-   HPLC high performance liquid chromatography-   IC₅₀ drug concentration required for 50% inhibition-   i.e. that is-   insol insoluble-   IPA isopropylamine-   IR infrared-   J coupling constant (NMR spectroscopy)-   LAH lithium aluminum hydride-   LC liquid chromatography-   LC-MS liquid chromatography-mass spectrometry-   LDA lithium diisopropylamide-   MAPK mitogen-activated protein kinase-   MeCN acetonitrile-   MEK MAPK/ERK kinase-   MHz megahertz-   min minute, minutes-   μL microliter-   mL milliliter-   μM micromolar-   mp melting point-   MS mass spectrum, mass spectrometry-   Ms methanesulfonyl-   m/z mass-to-charge ratio-   NBS N-bromosuccinimide-   nM nanomolar-   NMM 4-methylmorpholine-   obsd observed-   p page-   PBS phosphate buffered saline-   pp pages-   PdCl₂dppf    [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)-   Pd(OAc)₂ palladium acetate-   pH negative logarithm of hydrogen ion concentration-   pK negative logarithm of equilibrium constant-   pK_(a) negative logarithm of equilibrium constant for association-   PS-DIEA polystyrene-bound diisopropylethylamine-   q quartet (nmr)-   qt quintet (nmr)-   R_(f) retention factor (TLC)-   RT retention time (HPLC)-   rt room temperature-   TBAF tetra-n-butylammonium fluoride-   TBST tris buffered saline with tween-   TEA triethylamine-   THF tetrahydrofuran-   TFA trifluoroacetic acid-   TFFH fluoro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate-   TLC thin layer chromatography-   TMAD N,N,N′,N′-tetramethylethylenediamine-   TMSCl trimethylsilyl chloride-   Ts p-toluenesulfonyl-   v/v volume per volume-   w/v weight per volume-   w/w weight per weight-   Z zusammen (configuration)

General Procedures

In the subsequent paragraphs detailed general procedures for thesynthesis of key intermediates and compounds of the present inventionare described.

The schemes and procedures described below illustrate general syntheticroutes to the compounds of general formula (I) of the invention and arenot intended to be limiting. It is obvious to the person skilled in theart that the order of transformations as exemplified in the Schemes canbe modified in various ways. The order of transformations exemplified inthe Schemes is therefore not intended to be limiting. In addition,interconversion of any of the substituents, R1, R2, R3, R4 or R5 can beachieved before and/or after the exemplified transformations. Thesemodifications can be such as the introduction of protecting groups,cleavage of protecting groups, reduction or oxidation of functionalgroups, halogenation, metallation, substitution or other reactions knownto the person skilled in the art. These transformations include thosewhich introduce a functionality which allows for further interconversionof substituents. Appropriate protecting groups and their introductionand cleavage are well-known to the person skilled in the art (see forexample T. W. Greene and P. G. M. Wuts in Protective Groups in OrganicSynthesis, 3^(rd) edition, Wiley 1999). Specific examples are describedin the subsequent paragraphs.

A general route for the preparation of compounds of general formula (I)is described in Scheme 1.

A suitably substituted 1,3,5-trifluoro-2-nitrobenzene of general formula(G) is reacted with a suitably substituted 2-fluoro-aniline of generalformula (B), in a suitable solvent system, such as, for example, THF, inthe presence of a suitable base, such as, for example, lithiumhexamethyldisilazan at temperatures ranging from −78° C. to roomtemperature, preferably room temperature, to furnish3,5-difluoro-N-(2-fluorophenyl)-2-nitroaniline intermediates of generalformula (I).

Intermediates of general formula (I) are then converted to intermediatesof general formula (2) by reaction with a suitably substituted phenol ofgeneral formula (C), for example 3-amino phenol, in a suitable solventsystem, such as, for example, DMF, in the presence of a suitable base,such as, for example potassium carbonate, at a temperature between roomtemperature and the boiling point of the respective solvent.

Intermediates of general formula (2) are reacted with a suitablesulfonyl chloride of the general formula (D), for example ethyl sulfonylchloride, in the presence of a suitable base, such as, for examplepyridine, which optionally may function also as solvent, optionally in asuitable solvent such as, for example, dichloromethane, at temperaturesranging from 0° C. to room temperature, to furnish intermediates ofgeneral formula (3).

Intermediates of general formula (3) are then converted to intermediatesof general formula (4) by reaction with a suitable reducing agent agent,such as, for example, sodium dithionite, in a suitable solvent, such as,for example a THF/water mixture, at temperatures ranging from roomtemperature to the boiling point of the solvent. Alternative reagentsand conditions for reducing nitro groups are known in the art.

Intermediates of general formula (4) are then converted to compounds ofgeneral formula (I) by reaction with a suitable sulfonyl chloride of thegeneral formula (E), for example cyclopropane sulfonyl chloride, in thepresence of a suitable base, such as, for example, pyridine, whichoptionally may function also as solvent, optionally in a suitablesolvent, such as, for example, dichloromethane, at temperatures rangingfrom 0° C. to room temperature.

Alternatively, compounds of general formula (I) can be synthesisedaccording to the procedure depicted in Scheme 2. Compounds D, E and Hare commercially available or are prepared as described in the specificexperimental descriptions below.

Intermediates of general formula (1) are converted to intermediates ofgeneral formula (5) by reaction with a suitably protected substitutedphenol of general formula (H), for example tert-butyl(3-hydroxyphenyl)carbamate, in a suitable solvent system, such as, forexample, DMF, in the presence of a suitable base, such as, for examplecaesium carbonate, at a temperature between room temperature and theboiling point of the respective solvent.

Intermediates of general formula (5) are then converted to intermediatesof general formula (6) by reaction with a suitable reducing agent, suchas, for example sodium dithionite, in a suitable solvent, such as, forexample THF, at temperatures ranging from room temperature to theboiling point of the solvent.

Intermediates of general formula (6) are then converted to intermediatesof general formula (7) by reaction with a suitable sulfonyl chloride ofthe general formula (E), for example cyclopropane sulfonyl chloride, inthe presence of a suitable base, such as, for example, pyridine, in asuitable solvent, such as, for example, pyridine, at temperaturesranging from 0° C. to room temperature.

Intermediates of general formula (7) are converted to intermediates ofgeneral formula (8) by cleavage of the protecting group by means knownto the person skilled in the art, for example, cleavage of thetert-butoxycarbonyl (Boc) group in the presence of a suitable acid, suchas, for example, TFA, in a suitable solvent, such as, for example, DCM,at temperatures ranging from room temperature to the boiling point ofthe solvent.

Intermediates of general formula (8) are then reacted with a suitablesulfonyl chloride of the general formula (D), for example, isopropylsulfonyl chloride, in the presence of a suitable base, such as, forexample, pyridine, in a suitable solvent, such as, for example,pyridine, at temperatures ranging from 0° C. to room temperature, tofurnish compounds of general formula (I).

Alternatively, compounds of general formula (Ic) can be synthesisedaccording to the procedure depicted in Scheme 3. Compounds E and J arecommercially available.

A suitably substituted phenol of general formula (J), is converted tothe corresponding tert-butyloxy carbonyl (BOC) protected sulfamoylderivative of general formula (9) by reaction with chlorosulfonylisocyanate and tert. butanol in the presence of a suitable base, suchas, for example, triethylamine, at temperatures ranging from 0° C. toroom temperature, preferably room temperature [see for exampleTetrahedron 1993, 49, 65-76].

Intermediates of general formula (9) are converted to intermediates ofgeneral formula (10) by reaction with intermediates of general formula(I), in a suitable solvent system, such as, for example, DMF, in thepresence of a suitable base, such as, for example caesium carbonate, ata temperature between room temperature and the boiling point of therespective solvent.

Intermediates of general formula (10) are then converted tointermediates of general formula (11) by reaction with a suitablereduction agent, such as, for example, sodium dithionite, in a suitablesolvent, such as, for example, THF, at temperatures ranging from roomtemperature to the boiling point of the solvent.

Intermediates of general formula (11) are then converted tointermediates of general formula (12) by reaction with a suitablesulfonyl chloride of the general formula (E), for example, cyclopropanesulfonyl chloride, in the presence of a suitable base, such as, forexample, pyridine, in a suitable solvent, such as, for example,pyridine, at temperatures ranging from 0° C. to room temperature.

Intermediates of general formula (12) are converted to compounds ofgeneral formula (Ic) by cleavage of the protecting group by means knownto the person skilled in the art, for example, cleavage of thetert-butoxy carbonyl (Boc) group in the presence of a suitable acid,such as, for example, TFA, in a suitable solvent, such as, for exampleDCM, at temperatures ranging from room temperature to the boiling pointof the solvent.

Compounds of general formula (Ia) can be converted into compounds ofgeneral formula (Ib) according to the procedure depicted in Scheme 4.

Compounds of general formula (Ia) are converted to compounds of generalformula (Ib) by coupling reactions know to the person skilled in theart, preferably by a Sonogashira or Sonogashira-type coupling reactionwith acetylene or an acetylene equivalent (vide infra).

An iodo- or bromo-containing intermediate, for example of generalformula (Ia), can be reacted with acetylene in the presence of catalyticamounts of a Pd catalyst such as, for example, PdCl₂(PPh₃)₂, catalyticamounts of copper iodide, in the presence of a solvent such as DMF andoptionally in the presence of a base, such as, for example, a trialkylamine base, to form the corresponding alkyne derivative (Ib).Alternatively, mono-trialkylsilyl-protected acetylene such as forexample, trimethylsilyl (TMS) acetylene, may be employed in such aSonogashira-type coupling under conditions as described above followedby cleavage of the trialkylsilyl group by treatment with, for example,tetrabutylammonium fluoride or potassium carbonate in methanol.Alternatively, by using tetrabutylammonium fluoride as base in theSonogashira-type coupling, coupling of TMS acetylene and cleavage of theTMS-group can be achieved in a one pot transformation. Transitionmetal-catalyzed couplings of (hetero)aryl halides with alkynes andtrialkylsilyl alkynes are well known to the person skilled in the art(see for example (a) Chinchilla, R.; Najera, C. Chem. Rev. 2007, 107,874; (b) Negishi, E.-i., Anastasia, L. Chem. Rev. 2003, 103, 1979; seealso: (c) Eur. J. Org. Chem. 2005, 20, 4256; (d) J. Org. Chem. 2006, 71,2535 and references therein; (e) Chem. Commun. 2004, 17, 1934). Variouspalladium-catalyst/co-catalyst/ligand/base/solvent combinations havebeen published in the scientific literature which allow a fine-tuning ofthe required reaction conditions in order to allow for a broad set ofadditional functional groups on both coupling partners (see referencesin the above cited reviews). Additionally, recently developed proceduresemploying e.g. zinc acetylides, alkynyl magnesium salts or alkynyltrifluoroborate salts further broaden the scope of this process.

Specific Experimental Descriptions

NMR peak forms in the following specific experimental descriptions arestated as they appear in the spectra, possible higher order effects havenot been considered. Names of compounds were generated using ACD/NameBatch version 12.00. In some cases generally accepted names ofcommercially available reagents were used. Reactions employing microwaveirradiation may be run with a Biotage Initator® microwave ovenoptionally equipped with a robotic unit. The reported reaction timesemploying microwave heating are intended to be understood as fixedreaction times after reaching the indicated reaction temperature. Thecompounds and intermediates produced according to the methods of theinvention may require purification. Purification of organic compounds iswell known to the person skilled in the art and there may be severalways of purifying the same compound. In some cases, no purification maybe necessary. In some cases, the compounds may be purified bycrystallization. In some cases, impurities may be stirred out using asuitable solvent. In some cases, the compounds may be purified bychromatography, particularly flash column chromatography, using forexample prepacked silica gel cartridges, e.g. from Separtis such asIsolute® Flash silica gel or Isolute® Flash NH₂ silica gel incombination with a Flashmaster II autopurifier (Argonaut/Biotage) andeluents such as gradients of hexane/ethyl acetate or DCM/ethanol. Insome cases, the compounds may be purified by preparative HPLC using forexample a Waters autopurifier equipped with a diode array detectorand/or on-line electrospray ionization mass spectrometer in combinationwith a suitable prepacked reverse phase column and eluents such asgradients of water and acetonitrile which may contain additives such astrifluoroacetic acid or aqueous ammonia. In some cases, purificationmethods as described above can provide those compounds of the presentinvention which possess a sufficiently basic or acidic functionality inthe form of a salt, such as, in the case of a compound of the presentinvention which is sufficiently basic, a trifluoroacetate or formatesalt for example, or, in the case of a compound of the present inventionwhich is sufficiently acidic, an ammonium salt for example. A salt ofthis type can either be transformed into its free base or free acidform, respectively, by various methods known to the person skilled inthe art, or be used as salts in subsequent biological assays. It is tobe understood that the specific form (e.g. salt, free base etc) of acompound of the present invention as isolated as described herein is notnecessarily the only form in which said compound can be applied to abiological assay in order to quantify the specific biological activity.

The percentage yields reported in the following examples are based onthe starting component that was used in the lowest molar amount. Air andmoisture sensitive liquids and solutions were transferred via syringe orcannula, and introduced into reaction vessels through rubber septa.Commercial grade reagents and solvents were used without furtherpurification. The term “concentrated under reduced pressure” refers touse of a Buchi rotary evaporator at a minimum pressure of approximately15 mm of Hg. All temperatures are reported uncorrected in degreesCelsius (° C.).

In order that this invention may be better understood, the followingexamples are set forth. These examples are for the purpose ofillustration only, and are not to be to construed as limiting the scopeof the invention in any manner. All publications mentioned herein areincorporated by reference in their entirety.

In the subsequent paragraphs detailed general procedures for thesynthesis of key intermediates and compounds of the present inventionare described.

General Procedure 1 (GP 1): Sulfonamide formation

1. eq. of the respective amine were dissolved in pyridine (ca. 4 mL permmol amine) and treated with 1.2 to 2 eq. of the respective sulfonylchloride. The reaction mixture was stirred at rt until LCMS analysisshowed complete starting material consumption. Optionally, additionalaliquots of the respective sulfonyl chloride were added to complete theturnover. The reaction mixture was quenched with water and extractedseveral times with dichloromethane. The combined organic layers werewashed with brine, filtered and concentrated in vacuo to give the crudetarget compound. Preparative HPLC purification or flash columnchromatography provided the respective target compound.

Analytical LCMS Conditions A:

LCMS-data given in the subsequent specific experimental descriptionsrefer (unless otherwise noted) to the following conditions:

System: Waters Aqcuity UPLC-MS: Binary Solvent Manager, SampleManager/Organizer, PDA, ELSD, ZQ4000 Column: Aqcuity UPLC BEH C18 1.750x2.1 mm Solvent: A = H2O + 0.05% HCOOH B = Acetonitril + 0.05% HCOOHGradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B Flow: 0.8 mL/minTemperature: 60° C. Injection: 2.0 μl Detection: DAD scan range 210-400nm −> Peaktable ELSD −> Peaktable MS ESI+, ESI− Switch −> diverse scanranges possible (Report Header) scan range 100-1000 m/z scan range160-1000 m/z scan range 160-2000 m/z

Preparative HPLC Conditions B:

“Purification by preparative HPLC” in the subsequent specificexperimental descriptions refers to (unless otherwise noted) thefollowing conditions:

Analytics:

System: Waters Aqcuity UPLC-MS: Binary Solvent Manager, SampleManager/Organizer, Column Manager, PDA, ELSD, SQD 3001 Column: AqcuityBEH C18 1.7 50x2.1 mm Solvent: A = H₂O + 0.1% HCOOH B = AcetonitrilGradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B Flow: 0.8 mL/minTemperature: 60° C. Injection: 2.0 μl Detection: DAD scan range 210-400nm MS ESI+, ESI−, scan range 160-1000 m/z ELSD

Preparation:

System: Waters Autopurificationsystem: Pump 2545, Sample Manager 2767,CFO, DAD 2996, ELSD 2424, SQD 3001 Column: XBrigde C18 5 μm 100x30 mmSolvent: A = H₂O + 0.1% HCOOH B = Acetonitril Gradient: 0-1 min 1% B,1-8 min 1-99% B, 8-10 min 99% B Flow: 50 mL/min Temperature: RTSolution: Max. 250 mg/2.5 mL DMSO o. DMF Injection: 1 x 2.5 mLDetection: DAD scan range 210-400 nm MS ESI+, ESI−, scan range 160-1000m/z

Chiral HPLC Conditions C:

Chiral HPLC-data given in the subsequent specific experimentaldescriptions refer to the following conditions:

Analytics:

System: Dionex: Pump 680, ASI 100, Waters: UV-Detektor 2487 Column:Chiralpak IC 5 μm 150x4.6 mm Solvent: Hexan/Ethanol 80:20 + 0.1%Diethylamin Flow: 1.0 mL/min Temperature: 25° C. Solution: 1.0 mg/mLEtOH/MeOH 1:1 Injection: 5.0 μl Detection: UV 280 nm

Preparation:

System: Agilent: Prep 1200, 2xPrep Pump, DLA, MWD, Prep FC, ESA: CoronaColumn: Chiralpak IC 5 μm 250x30 mm Solvent: Hexan/Ethanol 80:20 + 0.1%Diethylamin Flow: 40 mL/min Temperature: RT Solution: 660 mg/5.6 mL EtOHInjection: 8 x 0.7 mL Detection: UV 280 nm

Flash Column Chromatography Conditions A

“Purification by (flash) column chromatography” as stated in thesubsequent specific experimental descriptions refers to the use ofBiotage Flashmaster II or Isolera (SP4) purification systems. Fortechnical specifications see “Biotage product catalogue” onwww.biotage.com.

Determination of Optical Rotation Conditions

Optical rotations were measured in DMSO, at 589 nm wavelength, 20° C.,concentration 1.0000 g/100 ml, intergration time 10 s, film thickness100.00 mm.

Synthetic Intermediates Intermediate 1.A Preparation of3,5-difluoro-N-(2-fluoro-4-iodophenyl)-2-nitroaniline

25 g 1,3,5-trifluoro-2-nitrobenzene (141.2 mmol, 1 eq.) and 33.5 g2-fluoro-4-iodoaniline (141.2 mmol. 1 eq.) were dissolved in 250 mL dryTHF and cooled with ice-salt to −10 to 0° C. upon which 424 mL lithiumhexamethyldisilazide (LiHMDS) solution (1M in THF; 424 mmol, 3. eq.)were added slowly over a 1 h periode. Upon completion of addition ofbase, the reaction mixtures was allowed to warm to rt and stirring wascontinued for 5 days. The reaction mixture was quenched with 0.5 Nhydrochloric acid and diluted with ethyl acetate. Saturated ammoniumchloride solution was added to facilitate phase separation. Theseparated aqueous phase was reextracted twice with ethyl acetate. Thecombined organic layers were washed with brine, dried over sodiumsulfate, filtered and concentrated in vacuo to give dark solid residue.The residue was purified by flash column chromatography on silica gelwith hexane and hexane/ethyl acetate 8:2 as eluent to yield 45.8 gram(82% yield) of the analytically pure target compound as a slightlybrownish solid.

¹H-NMR (d₆-DMSO; 400 MHz): δ=8.86 (s, 1H); 7.73 (dd, 1H); 7.55 (dd, 1H);7.13 (t, 1H); 6.96 (ddd, 1H); 6.40-6.44 (m, 1H).

LC-MS: retention time: 1.5 min

MS ES⁺: 392.9 [M−H]⁻

Intermediate 2.A Preparation of3-(3-aminophenoxy)-5-fluoro-N-(2-fluoro-4-iodophenyl)-2-nitroaniline

18.4 g 3,5-difluoro-N-(2-fluoro-4-iodophenyl)-2-nitroaniline (46.9mmol, 1. eq.), 5.6 g 3-amino phenol (51.6 mmol. 1.1 eq.) and 9.7 gpotassium carbonate (70.3 mmol, 1.5 eq.) were weighed into around-bottom flask. 300 mL dry DMF were added and the resulting slurrywas stirred at rt for 3 days. The reaction mixture was quenched byaddition of water and diluted with ethyl acetate and phases wereseparated. The separated aqueous layer was reextracted twice with ethylacetate. The combined organic layers were washed with brine, dried oversodium sulfate, filtered and concentrated in vacuo. The residue waspurified by flash column chromatography on silica gel with a gradientfrom pure hexane to hexane/ethyl acetate 1:1 as eluent to yield 11.6 g(51% yield) of the target compound which contained a minor amount of theregioisomeric5-(3-aminophenoxy)-3-fluoro-N-(2-fluoro-4-iodophenyl)-2-nitroaniline. Ingeneral, this regioisomeric mixture was moved forward through thesubsequent transformations and separation into the pure regioisomericexample compounds was done by preparative HPLC after the finaltransformations (see below).

¹H-NMR (d₆-DMSO; 300 MHz): δ=8.45 (s, 1H); 7.68 (dd, 1H); 7.52 (dd, 1H);7.07 (t, 1H); 7.01 (t, 1H); 6.38 (dd, 1H); 6.26-6.31 (m, 1H); 6.16-6.24(m, 3H); 5.34 (s, 2 H).

LC-MS: retention time: 1.56 min

MS ES⁺: 484.20 [M+H]⁺

Intermediate 3.A Preparation ofN-(3-{5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-nitrophenoxy}-phenyl)ethanesulfonamide

11.7 g3-(3-aminophenoxy)-5-fluoro-N-(2-fluoro-4-iodophenyl)-2-nitroaniline(24.3 mmol, 1. eq.; containing a minor amount of the regioisomeric5-(3-aminophenoxy)-3-fluoro-N-(2-fluoro-4-iodophenyl)-2-nitroaniline)was dissolved in 98 mL pyridine, set under an atmosphere of nitrogen andtreated sequentially with 3.4 mL ethyl sulfonyl chloride (36.4 mmol, 1.5eq.; dissolved in pyridine). The resulting reaction mixture was stirredat rt for 20 h after which LCMC analysis showed final turnover. Thereaction mixture was quenched by addition of water and diluted withethyl acetate and phases were separated. The separated aqueous layer wasreextracted twice with ethyl acetate. The combined organic layers werewashed with brine, dried over sodium sulfate, filtered and concentratedin vacuo to give 14.8 g of the crude target compound (which contained aminor amount of the regioisomericN-(3-{3-fluoro-5-[(2-fluoro-4-iodophenyl)amino]-2-nitrophenoxy}-phenyl)ethanesulfonamide)which was used without further purification in the subsequenttransformations.

¹H-NMR (d₆-DMSO; 300 MHz): δ=9.97 (s, 1H); 8.52 (s, 1H); 7.70 (dd, 1H);7.52 (br. d, 1H); 7.34 (t, 1H); 7.10 (t, 1H); 7.04 (dd, 1H); 6.92 (s,1H); 6.79 (dd, 1H); 6.34-6.39 (m, 2H); 3.10 (q, 2H); 1.15 (t, 3H).

LC-MS: retention time: 1.49 min

MS ES⁺: 576.01 [M+H]

Intermediate 4.A Preparation ofN-(3-{2-amino-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}-phenyl)ethanesulfonamide

14.8 g crudeN-(3-{5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-nitrophenoxy}-phenyl)ethanesulfonamide(Intermediate 3.A) (25.9 mmol, 1. eq.; containing a minor amount of theregioisomericN-(3-{3-fluoro-5-[(2-fluoro-4-iodophenyl)amino]-2-nitrophenoxy}-phenyl)ethanesulfonamide)was dissolved in 560 mL THF, heated to 50° C. (bath temperature) andtreated within 30 minutes with a solution of 76 g sodium dithionite (440mmol, 17 eq.) in 420 mL water. Stirring at 50° C. bath temperature wascontinued for 150 min upon which LCMS analysis showed complete turnover.The reaction mixture was cooled to rt, THF was removed in vacuo and theresidue was partitioned between saturated sodium bicarbonate solutionand ethyl acetate. After phase separation, the separated aqueous layerwas reextracted twice with ethyl acetate. The combined organic layerswere washed with brine, dried over sodium sulfate, filtered andconcentrated in vacuo to give the crude target compound. Flash columnchromatography on silica gel with a hexane/methyl-tert-butyl ethergradient as eluent provided 11.8 g of the target compound (21.6 mmol,84% yield), which contained a minor amount of the regioisomericN-(3-{2-amino-3-fluoro-5-[(2-fluoro-4-iodophenyl)amino]phenoxy}-phenyl)ethanesulfonamide.

¹H-NMR (d₆-DMSO; 300 MHz): δ=9.85 (br. s, 1H); 7.52 (dd, 1H); 7.42 (s,1H); 7.31 (br. d, 1H); 7.25 (t, 1H); 6.90 (dd, 1H); 6.84 (t, 1H);6.55-6.65 (m, 3H); 6.48 (dd, 1H); 4.47 (s, 2H); 3.07 (q, 2H); 1.14 (t,3H).

LC-MS: retention time: 1.46 min

MS ES⁺: 546.14 [M+H]⁺

Intermediate 5.A Preparation of3-(3-amino-2-methylphenoxy)-5-fluoro-N-(2-fluoro-4-iodophenyl)-2-nitroaniline

3.6 g of 3,5-difluoro-N-(2-fluoro-4-iodophenyl)-2-nitroaniline(Intermediate 1A; 9.2 mmol, 1 eq.), 1.1 g 3-amino-2-methyl phenol (9.2mmol, 1 eq.) and 4.5 g caesium carbonate (13.8 mmol, 1.5 eq.) weresuspended in 35 mL DMF and stirred at rt for 16 h. LCMS analysis showedcomplete turnover. The reaction mixture was diluted with ethyl acetate,quenched with saturated sodium chloride solution, the layers wereseparated and the aqueous layer was reextracted with ethyl acetate. Thecombined organic layers were dried, filtered and concentrated in vacuo.The crude product was purified by flash column chromatography(n-hexane/ethyl acetate gradient) providing 1.18 of the desired product(26% yield).

¹H-NMR (d₆-DMSO; 400 MHz): δ=8.44 (s, 1H); 7.69 (dd, 1H); 7.52 (dd, 1H);7.08 (t, 1H); 6.92 (t, 1H); 6.53 (d, 1H); 6.19-6.24 (m, 2H); 5.84 (dd,1H); 5.17 (s, 2H); 1.84 (s, 3H).

LC-MS: retention time: 1.67 min

MS ES⁺: 497.8 [M+H]⁺

Intermediate 6.A Preparation ofN-(3-{5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-nitrophenoxy}-2-methylphenyl)ethanesulfonamide

1.18 g3-(3-amino-2-methylphenoxy)-5-fluoro-N-(2-fluoro-4-iodophenyl)-2-nitroaniline(2.3 mmol, 1 eq.) were dissolved in 9.6 mL pyridine, cooled to 0° C.,treated with 292 μL ethyl sulfonyl chloride (3.1 mmol, 1.2 eq.) andstirred at 0° C. for 3 hours. The reaction mixture was diluted withtoluene and concentrated in vacuo. The residue was partitioned betweenwater and ethyl acetate. The aqueous layer was reextracted with ethylacetate. The combined organic layers were dried, filtered andconcentrated in vacuo to yield 1.6 g of the crude product, which wastaken up in toluene and again concentrated in vacuo. The crude productwas used in the subsequent transformations without further purification.

¹H-NMR (d₆-DMSO; 300 MHz): δ=9.25 (br. s, 1H); 8.51 (s, 1H); 7.70 (dd,1H); 7.52 (dd, 1H); 7.05-7.26 (3H; obscured by residual toluene); 6.95(dd, 1H); 6.26-6.31 (m, 1H); 5.99 (dd, 1H); 3.09 (q, 2H); 2.12 (s, 3H);1.22 (t, 3H).

LC-MS: retention time: 1.48 min

MS ES″: 587.9 [M−H]⁻

Intermediate 7.A Preparation ofN-(3-{2-amino-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}-2-methylphenyl)ethanesulfonamide

1.6 g crudeN-(3-{5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-nitrophenoxy}-2-methylphenyl)-ethanesulfonamide(2.7 mmol, 1 eq.) were dissolved in 40 mL THF/EtOH 1:1 and treated with3.1 g stannous chloride dihydrate (SnCl₂ 2H₂O) (13.6 mmol, 5 eq.) andthe resulting mixture was refluxed for 16 h. The reaction mixture wasconcentrated, the residue quenched with 26% NH₃ and extracted with DCM.The combined organic layers were washed with ammonium chloride solutionand saturated sodium chloride solution, dried, filtered and concentratedin vacuo. The residue was purified by flash column chromatography togive 996 mg of the target compound (65% yield).

¹H-NMR (d₆-DMSO; 300 MHz): δ=9.18 (br. s, 1H); 7.51 (dd, 1H); 7.40 (s,1H); 7.32 (br. d, 1H); 7.15 (t, 1H); 7.06 (d, 1H); 6.68 (dd, 1H); 6.61(t, 1H); 6.51 (dd, 1H); 6.18 (dd, 1H); 4.51 (s, 2H); 3.07 (q, 2H); 2.19(s, 3H); 1.23 (t, 3H).

LC-MS: retention time: 1.47 min

MS ES⁺: 560.14 [M+H]⁺

Intermediate 8.A Preparation of tert-butyl(3-{5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-nitrophenoxy}phenyl)carbamate

3.0 g of 3,5-difluoro-N-(2-fluoro-4-iodophenyl)-2-nitroaniline(Intermediate 1A; 70% pure, 5.3 mmol, 1 eq.), 1.1 g N-Boc-3-amino-phenol(5.3 mmol, 1 eq.) and 3.4 g caesium carbonate (10.7 mmol, 1.5 eq.) weresuspended in 30 mL DMF and stirred at rt for 16 h. LCMS analysis showedcomplete turnover. The reaction mixture was partitioned between waterand ethyl acetate, the layers were separated and the aqueous layer wasreextracted with ethyl acetate. The combined organic layers were dried,filtered and concentrated in vacuo. The crude product was purified byflash column chromatography (n-hexane/ethyl acetate gradient) providing1.35 of the desired product (44% yield).

¹H-NMR (d₆-DMSO; 300 MHz): δ=9.50 (s, 1H); 8.48 (s, 1H); 7.69 (dd, 1H);7.52 (dd, 1H); 7.22-7.32 (m, 3H); 7.09 (t, 1H); 6.69 (dt, 1H); 6.30-6.36(m, 1H); 6.26 (dd, 1H); 1.43 (s, 9H).

LC-MS: retention time: 1.68 min

MS ES⁻: 582.0 [M−H]⁻

Intermediate 9.A Preparation of tert-butyl(3-{2-amino-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}phenyl)carbamate

1.35 g of tert-butyl(3-{5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-nitrophenoxy}phenyl)carbamate(2.3 mmol, 1 eq.) were dissolved in 43 mL THF and heated to 50° C. uponwhich a solution of 6.8 g sodium dithionite (39 mmol, 17 eq.) in 35 mLwater was added and stirring was continued at that temperature for 1 h.The layers were separated and the aqueous layer was reextracted withethyl acetate. The combined organic layers were dried, filtered andconcentrated in vacuo. The crude product (1.5 g) was used withoutfurther purification.

¹H-NMR (d₆-DMSO; 300 MHz): δ=9.39 (s, 1H); 7.51 (dd, 1H); 7.41 (s, 1H);7.31 (d, 1H); 7.09-7.23 (m, 3H); 6.52-6.62 (m, 3H); 6.41 (dd, 1H); 4.42(s, 2H); 1.42 (s, 9 H).

LC-MS: retention time: 1.62 min

MS ES⁺: 553.9 [M+H]⁺

Intermediate 10.A Preparation of tert-butyl(3-{2-[(cyclopropylsulfonyl)amino]-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}phenyl)carbamate

1.5 g of crude tert-butyl(3-{2-amino-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}phenyl)carbamate(2.7 mmol, 1 eq.) were dissolved in 10 mL pyridine and 457 mgcyclopropyl sulfonyl chloride (3.3 mmol, 1.2 eq.) and stirring wascontinued at that temperature for 16 h. The reaction mixture was dilutedwith toluene and concentrated in vacuo. The residue was partitionedbetween DCM and water, the aqueous layer was reextracted with DCM. Thecombined organic layers were dried, filtered and concentrated in vacuo.The crude product was purified by flash column chromatography yielding1.19 g of the target compound (67% yield).

¹H-NMR (d₆-DMSO; 300 MHz): δ=9.47 (s, 1H); 9.11 (br. s, 1H); 7.64 (dd,1H); 7.57 (s, 1H); 7.44 (d, 1H); 7.23-7.31 (m, 3H); 7.12 (t, 1H);6.68-6.73 (m, 1H); 6.55 (dd, 1H); 6.04 (dd, 1H); 2.60-2.70 (m, 1H); 1.42(s, 9H); 0.80-0.90 (m, 4H).

LC-MS: retention time: 1.63 min

MS ES⁻: 655.9 [M−H]⁻

Intermediate 11.A Preparation ofN-{2-(3-aminophenoxy)-4-fluoro-6-[(2-fluoro-4-iodophenyl)amino]phenyl}cyclopropanesulfonamide

1.19 g of tert-butyl(3-{2-[(cyclopropylsulfonyl)amino]-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}phenyl)carbamate(1.8 mmol, 1 eq.) were dissolved in 15 mL DCM, treated with 2 mL TFA andstirred at rt for 6 h. The reaction mixture was cooled to 0° C. andadjusted with 1N Sodium hydroxide to pH 10. The layers were separated,the aqueous layer was reextracted with DCM. The combined organic layerswere dried, filtered and concentrated in vacuo to yield 776 mg of thecrude product (77% yield), which was used without further purification.

¹H-NMR (d₆-DMSO; 400 MHz): δ=9.08 (br. s, 1H); 7.63 (dd, 1H); 7.53 (s,1H); 7.44 (d, 1H); 7.11 (t, 1H); 7.02 (t, 1H); 6.53 (dd, 1H); 6.38 (dd,1H); 6.28 (t, 1H); 6.22 (dd, 1H); 6.03 (dd, 1H); 5.29 (s, 2H); 2.60-2.67(m, 1H); 0.81-0.91 (m, 4H).

LC-MS: retention time: 1.46 min

MS ES⁺: 557.8 [M+H]⁺

Intermediate 12.A Preparation of3-(3-amino-4-fluorophenoxy)-5-fluoro-N-(2-fluoro-4-iodophenyl)-2-nitroaniline

1 g of 3,5-difluoro-N-(2-fluoro-4-iodophenyl)-2-nitroaniline(Intermediate 1A; 2.537 mmol, 1 eq.), 194 mg 3-amino-4-fluorophenol(1.522 mmol, 0.6 eq.) and 1.240 g caesium carbonate (3.806 mmol, 1.5eq.) were suspended in 12 mL DMF and stirred at rt for 72 h. Thereaction mixture was partitioned between butan-2-one and half saturatedbrine. The layers were separated and the aqueous layer was reextractedtwice with ethyl butan-2-one. The combined organic layers were washedwith brine, dried, filtered and concentrated in vacuo. The crude productwas purified by flash column chromatography (n-hexane/ethyl acetategradient) providing 450 mg of a mixture of the desired product with itspara regioisomer that eluted with the same retention time (63% UVpurity). This material was used without further purification.

LC-MS: retention time: 1.54 min

MS ES⁺: 502.39 [M+H]⁺

Intermediate 13.A Preparation ofN-(2-fluoro-5-{5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-nitrophenoxy}phenyl)sulfuricdiamide

Solution A: 254 mg chlorsulfonyl isocyanate (1.797 mmol, 2 eq.) weredissolved in 0.427 mL DCM, the mixture heated to 40° C. and then treatedwith formic acid and treated with 71 μL formic acid (1.887 mmol, 2.1eq.) and 1.3 μl DMA (0.014 mmol, 0.016 eq.) dropwise while gas evolutionoccurred. The mixture was heated to reflux for another 15 minutes.

450 mg crude3-(3-amino-4-fluorophenoxy)-5-fluoro-N-(2-fluoro-4-iodophenyl)-2-nitroaniline(0.898 mmol, 1 eq.) were dissolved in 3.5 mL DMA, and treated with 235μL DIPEA (1.348 mmol, 1.5 eq.). Upon cooling to 0° C., solution A wasadded dropwise and the resulting mixture was stirred at rt for 18 h. Thereaction mixture was partitioned between water and ethyl ether and theaqueous layer was reextracted twice with ethyl ether. The combinedorganic layers were washed once with half saturated brine, once withbrine, dried, filtered and concentrated in vacuo, providing 597 mg ofcrude product containing a mixture of the target compound and its pararegioisomer (71% ortho, 11% para) which was used in the subsequenttransformations without further purification.

LC-MS: retention time: 1.40 min

MS ES⁺: 581.27 [M+H]⁺

Intermediate 14.A Preparation ofN-(5-{2-amino-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}-2-fluorophenyl)sulfuricdiamide

597 mg crudeN-(2-fluoro-5-{5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-nitrophenoxy}phenyl)sulfuricdiamide (1.028 mmol, 1 eq.) were dissolved in 20 mL THF, warmed to 50°C., treated with a solution of 3.044 g sodium dithionite (17.484 mmol,17 eq.) in 16 ml of water dropwise over a period of 30 minutes. Theresulting mixture was stirred at this temperature for another 5 h. Uponcooling to rt the phases separated. The organic layer was separated,concentrated in vacuo and redissolved in ethyl acetate. The aqueouselayer was diluted with saturated sodium bicarbonate solution and thenextracted twice with ethyl acetate. The combined organic layers werewashed once with half saturated brine and once with brine saturated,dried, filtered and concentrated in vacuo, providing 547 mg of crudeproduct which was used in the subsequent transformations without furtherpurification.

LC-MS: retention time: 1.34 min

MS ES⁺: 551.41 [M+H]⁺

Intermediate 15.A Preparation of tert-butyl[(3-hydroxybenzyl)sulfamoyl]carbamate

Solution A: 6,321 g chlorosulfonyl isocyanate (44.659 mmol, 1.1 eq.)were dissolved in 60 ml dry dichloromethane. A solution of tert. butanolin 30 ml dry dichloromethane was added at rt and stirred for another 5min.

Solution B: 5 g 3-(aminomethyl)phenol (40.599 mmol, 1 eq.) weresuspended in 110 mL dry dichloromethane, 6,791 ml triethylamine (48.719mmol, 1.2 eq.) were added and the mixture was cooled to 0° C. upon whichsolution A was added dropwise. Stirring at rt was continued for 1 h.

The reaction mixture was quenched by addition of half concentratedammonium chloride solution, diluted with dichloromethane and phases wereseparated. The separated aqueous phase was reextracted twice withdichloromethane. The combined organic layers were washed with brine,dried over sodium sulfate, filtered and concentrated in vacuo to givethe crude product. The residue was purified by crystallization to yield6.469 gram (53% yield) of the target compound.

¹H-NMR (d₆-DMSO; 300 MHz): δ=10.79 (br. s, 1H); 9.30 (s, 1H); 8.04 (dd,1H); 7.05 (dd, 1H); 6.72-6.65 (m, 2H); 6.59 (dm, 1H); 3.89 (d, 2H); 1.37(s, 9H).

LC-MS: retention time: 0.88 min

MS ES⁻: 301.2 [M−H]⁻

Intermediate 16.A Preparation of tert-butyl[(3-{5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-nitrophenoxy}benzyl)sulfamoyl]carbamate

2.0 g of 3,5-difluoro-N-(2-fluoro-4-iodophenyl)-2-nitroaniline(Intermediate 1A; 64% pure, 3.25 mmol, 1 eq.), 0.98 g tert-butyl[(3-hydroxybenzyl)sulfamoyl]carbamate (Intermediate 2A; 3.24 mmol, 1eq.) and 1.6 g caesium carbonate (4.9 mmol, 1.5 eq.) were suspended in25 mL DMF and stirred in a closed vial at rt for 3 days. Additional 0.4eq. tert-butyl [(3-hydroxybenzyl)sulfamoyl]carbamate and 0.4 eq. caesiumcarbonate were added and stirring was continued for 2 days. The reactionmixture was partitioned between sodium chloride solution and ether, thelayers were separated and the aqueous layer was reextracted with ether.The combined organic layers were dried, filtered and concentrated invacuo to yield 3.08 g of the crude product (as a regioisomeric mixturecontaining some unreacted starting material), which was used in thesubsequent reaction without further purification.

LC-MS: retention time: 1.55 min

MS ES⁻: 674.8 [M−H]⁻

Intermediate 17.A Preparation of tert-butyl[(3-{2-amino-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}benzyl)sulfamoyl]carbamate

3.08 g of tert-butyl[(3-{5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-nitrophenoxy}benzyl)sulfamoyl]carbamate(4.55 mmol, 1 eq.) were dissolved in 88 mL THF and heated to 50° C. uponwhich a solution of 13.5 g sodium dithionite (77.4 mmol, 17 eq.) in 70mL water was added and stirring was continued at that temperature for 5h. The layers were separated and the THF layer was concentrated invacuo. The residue was taken up in ethyl acetate, washed with sodiumbicarbonate solution and sodium chloride solution, dried, filtered andconcentrated in vacuo. The residue was further purified by flash columnchromatography (hexan to hexan/ethyl acetate 1:1 gradient) to yield 886mg (30% yield) of the target compound (as a 2-3:1 regioisomericmixture).

LC-MS: retention time: 1.51 min

MS ES⁺: 646.9 [M+H]⁺

Intermediate 18.A Preparation of tert-butyl[(3-{2-[(cyclopropylsulfonyl)amino]-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}benzyl)sulfamoyl]carbamate

864 mg of crude tert-butyl[(3-{2-amino-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}benzyl)sulfamoyl]carbamate(1.37 mmol, 1 eq.) were dissolved in 13 mL pyridine and 250 mgcyclopropyl sulfonyl chloride (1.78 mmol, 1.3 eq.) and stirring wascontinued at that temperature for 16 h. The reaction mixture was dilutedwith toluene and concentrated in vacuo. The residue was partitionedbetween ethyl acetate and water, the aqueous layer was reextracted withethyl acetate. The combined organic layers were dried, filtered andconcentrated in vacuo. The residue was further purified by flash columnchromatography (hexan to hexan/ethyl acetate 1:1 gradient) to yield 814mg of the target compound (70% UV-purity), which was used for thesubsequent transformation.

LC-MS: retention time: 1.52 min

MS ES⁻: 748.8 [M−H]⁻

EXAMPLE COMPOUNDS Example 1 Preparation ofN-(2-{3-[(ethylsulfonyl)amino]phenoxy}-4-fluoro-6-[(2-fluoro-4-iodophenyl)amino]phenyl)cyclopropanesulfonamide

154 mgN-(3-{2-amino-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}-phenyl)ethanesulfonamide(Intermediate 4.A) (0.28 mmol, 1. eq.; containing a minor amount of theregioisomericN-(3-{2-amino-3-fluoro-5-[(2-fluoro-4-iodophenyl)amino]phenoxy}-phenyl)ethanesulfonamide)was dissolved in 1 mL pyridine and treated with 79.4 mgcyclopropanesulfonyl chloride (0.565 mmol, 2 eq.). The reaction mixturewas stirred at rt for 48 h. The reaction mixture was quenched with waterand extracted several times with dichloromethane. The combined organiclayers were washed with brine, filtered and concentrated in vacuo togive the crude target compound. Preparative HPLC purification provided62 mg of the analytically pure target compound (0.09 mmol, 32% yield).

¹H-NMR (d₆-DMSO; 300 MHz): δ=9.92 (br. s, 1H); 9.09 (br. s, 1H); 7.64(dd, 1H); 7.59 (br. s, 1H); 7.45 (br. d, 1H); 7.34 (t, 1H); 7.12 (t,1H); 7.00-7.04 (m, 1H); 6.96 (t, 1H); 6.81 (dd, 1H); 6.57 (dd, 1H); 6.15(dd, 1H); 3.10 (q, 2H); 2.63-2.71 (m, 1H); 1.15 (t, 3H); 0.82-0.88 (m,4H).

LC-MS: retention time: 1.51 min

MS ES⁺: 650.17 [M+H]⁺

The following example compounds 2 to 5 were prepared in analogy toexample compound 1 by treatingN-(3-{2-amino-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}-phenyl)-ethanesulfonamide(Intermediate 4.A) with the respective commercially available sulfonylchlorides in the presence of pyridine.

Example Structure Name Analytical Data 2

N-(3-{5-fluoro-3-[(2- fluoro-4- iodophenyl)amino]-2- [(methylsulfonyl)-amino]phenoxy}- phenyl)ethane- sulfonamide ¹H-NMR (d₆-DMSO; 300 MHz): δ= 9.92 (br. s, 1 H); 9.12 (br. s, 1 H); 7.65 (dd, 1 H); 7.59 (s, 1 H);7.45 (br. d, 1 H); 7.34 (t, 1 H); 7.11 (t, 1 H); 7.00-7.04 (m, 1 H);6.96 (t, 1 H); 6.82 (dd, 1 H); 6.53 (dd, 1 H); 6.14 (dd, 1 H); 3.10 (q,2 H); 3.00 (s, 3 H); 1.15 (t, 3 H). LC-MS: retention time: 1.39 min MSES⁺: 623.7 [M + H]⁺ 3

N-(3-{2- [(ethylsulfonyl)amino]- 5-fluoro-3-[(2- fluoro-4-iodophenyl)amino]- phenoxy}phenyl)- ethanesulfonamide ¹H-NMR (d₆-DMSO;300 MHz): δ = 9.93 (br. s, 1 H); 9.08 (br. s, 1 H); 7.65 (dd, 1 H); 7.58(s, 1 H); 7.44 (br. d, 1 H); 7.34 (t, 1 H); 7.09 (t, 1 H); 7.02 (dd, 1H); 6.95 (t, 1 H); 6.80 (dd, 1 H); 6.58 (dd, 1 H); 6.17 (dd, 1 H); 3.10(br. q, 4 H); 1.19 (t, 3 H); 1.15 (t, 3 H). LC-MS: retention time: 1.43min MS ES⁺: 637.8 [M + H]⁺ 4

N-(2-{3- [(ethylsulfonyl)amino] phenoxy}-4-fluoro-6- [(2-fluoro-4-iodophenyl)amino]- phenyl)propane-2- sulfonamide ¹H-NMR (d₆-DMSO; 300MHz): δ = 9.94 (br. s, 1 H); 9.02 (br. s, 1 H); 7.65 (dd, 1 H); 7.56 (s,1 H); 7.44 (br. d, 1 H); 7.34 (t, 1 H); 7.09 (t, 1 H); 7.02 (dd, 1 H);6.94 (t, 1 H); 6.80 (dd, 1 H); 6.64 (dd, 1 H); 6.19 (dd, 1 H); 3.25 (m,1 H); 3.10 (q, 2 H); 1.24 (d, 6 H); 1.15 (t, 3 H). LC-MS: retentiontime: 1.46 min MS ES⁺: 651.9 [M + H]⁺ 5

N-(2-{3- [(ethylsulfonyl)amino] phenoxy}-4-fluoro-6- [(2-fluoro-4-iodophenyl)amino]- phenyl)cyclobutane- sulfonamide ¹H-NMR (d₆-DMSO; 300MHz): δ = 9.94 (br. s, 1 H); 9.01 (br. s, 1 H); 7.65 (dd, 1 H); 7.58 (s,1 H); 7.44 (br. d, 1 H); 7.35 (t, 1 H); 7.09 (t, 1 H); 7.03 (dd, 1 H);6.97 (t, 1 H); 6.83 (dd, 1 H); 6.59 (dd, 1 H); 6.13 (dd, 1 H); 3.92 (m,1 H); 3.11 (q, 2H); 2.09-2.32 (m, 4H); 1.76-1.88 (m, 2 H); 1.15 (t, 3H). LC-MS: retention time: 1.49 min MS ES⁺: 663.8 [M + H]⁺

Example 6 Preparation ofN-(3-{5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-(sulfamoylamino)phenoxy}phenyl)ethanesulfonamide

244 mgN-(3-{2-amino-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}-phenyl)ethanesulfonamide(0.448 mmol, 1 eq.) were dissolved in 10 mL DCM, treated with 0.781 mLDIPEA (4.5 mmol, 10 eq.) and 388 mg sulfamoyl chloride (3.36 mmol, 7.5eq.) and the resulting solution stirred at rt for four days. Thereaction mixture was portioned between ammonium chloride solution andDCM, the aqueous layer reextracted with DCM, the combined organic layersdried, filtered and concentrated in vacuo. Flash column chromatographyprovided 97 mg of the target compound (35% yield). A second productfraction was further purified by thick layer chromatography(hexane/ethyl acetate 1:1 as eluent) yielding additional 67 mg of thetarget compound (22% yield).

¹H-NMR (d₆-DMSO; 400 MHz): δ=9.94 (br. s, 1H); 8.58 (br. s, 1H);7.60-7.63 (m, 2H); 7.46 (br. d, 1H); 7.32 (t, 1H); 7.14 (t, 1H);6.96-7.02 (m, 4H); 6.82 (dd, 1H); 6.48 (dd, 1H); 6.01 (dd, 1H); 3.10 (q,2H); 1.16 (t, 3H).

LC-MS: retention time: 1.37 min

MS ES⁺: 624.7 [M+H]⁺

Example 7 Preparation ofN-(2-{3-[(ethylsulfonyl)amino]-2-methylphenoxy}-4-fluoro-6-[(2-fluoro-4-iodophenyl)amino]phenyl)cyclopropanesulfonamide

185 mgN-(3-{2-amino-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}-2-methylphenyl)ethanesulfonamide(0.33 mmol, 1 eq.) were dissolved in 3 mL pyridine, and treated with 49mg cyclopropyl sulfonyl chloride (0.33 mmol, 1 eq.) and stirred at itfor 16 h. Further 15 mg cyclopropyl sulfonyl chloride were added andstirring was continued for 4 h. Additional 15 mg cyclopropyl sulfonylchloride were added and stirring was continued overnight. The reactionmixture was quenched with water, diluted with toluene and concentratedin vacuo. The residue was portioned between water and ethyl acetate, theaqueous layer reextracted with ethyl acetate, the combined organiclayers dried, filtered and concentrated in vacuo. Flash columnchromatography provided 150 mg of the target compound (64% yield).

¹H-NMR (d₆-DMSO; 300 MHz): δ=9.16 (br. s, 2H); 7.62-7.65 (m, 2H); 7.45(br. d, 1H); 7.11-7.24 (m, 3H); 6.90 (d, 1H); 6.51 (dd, 1H); 5.78 (dd,1H); 3.10 (q, 2 H); 2.67-2.70 (m, 1H); 2.15 (s, 3H); 1.23 (t, 3H);0.83-0.86 (m, 4H).

LC-MS: retention time: 1.47 min

MS ES⁺: 663.8 [M+H]⁺

Example 8 Preparation ofN-(3-{5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-(sulfamoylamino)phenoxy}-2-methylphenyl)ethanesulfonamide

Solution A: 65 mg Chlorsulfonylisocyanate were dissolved in 0.11 mL DCM,heated to reflux and treated with 18 μL formic acid and 350 μL DMA.

140 mgN-(3-{2-amino-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}-2-methylphenyl)ethanesulfonamide(0.233 mmol, 1 eq.) were dissolved in 0.79 mL DMA, and treated with 61μL DIPEA. Solution A was added by syringe and the resulting mixture wasstirred at rt for 16 h. The reaction mixture was portioned between waterand ethyl acetate, the aqueous layer reextracted with ethyl acetate, thecombined organic layers dried, filtered and concentrated in vacuo. Flashcolumn chromatography provided 53 mg of the target compound (34% yield).

¹H-NMR (d₆-DMSO; 400 MHz): δ=9.15 (br. s, 1H); 8.59 (br. s, 1H);7.64-7.68 (m, 2H); 7.46 (br. d, 1H); 7.12-7.24 (m, 3H); 7.05 (s, 2H);6.94 (d, 1H); 6.43 (dd, 1H); 5.64 (dd, 1H); 3.10 (q, 2H); 2.16 (s, 3H);1.24 (t, 3H).

LC-MS: retention time: 1.35 min

MS ES⁻: 636.9 [M−H]⁻

Example 9 Preparation ofN-(2-{3-[(ethylsulfonyl)amino]phenoxy}-6-[(4-ethynyl-2-fluorophenyl)amino]-4-fluorophenyl)cyclopropanesulfonamide

Step 1

100 mgN-(2-{3-[(ethylsulfonyl)amino]phenoxy}-4-fluoro-6-[(2-fluoro-4-iodophenyl)-amino]phenyl)cyclopropanesulfonamide(0.154 mmol, 1 eq.), 3.5 mg Pd(dba)₂ (0.006 mmol; 4 mol %), 1.2 mgcopper (I) iodide (0.006 mmol; 4 mol %) and 8 mg triphenylphosphine(0.03 mmol; 20 mol %) are weighed into a microwave vial, dissolved in1.5 mL triethylamine, set under an argon atmosphere and supplied with128 μL trimethylsilyl acetylene (0.924 mmol, 6 eq.). The vial was cappedand heated to 60° C. for 3 h upon which LCMS analysis showed completeturnover. The reaction mixture was concentrated in vacuo and the crudeproduct was used without further purification.

LC-MS: retention time: 1.61 min

MS ES⁻: 618.0 [M−H]⁻

Step 2

The crude product from step 1 was dissolved in 2 mL THF and treated with0.24 mL TBAF solution (1.0 M in THF; 0.24 mmol, 1.55 eq.) and stirred atrt for 16 h. The reaction mixture was diluted with ethyl acetate, washedwith sat. sodium bicarbonate solution and subsequently with sat. sodiumchloride solution, dried and filtered. After concentration in vacuo theresidue was purified by preparative thin layer chromatography (5 DCplates 20×20 cm; hexane/ethyl acetate 1:1 as eluent) yielding 26 mg ofthe target compound (31% yield over 2 steps).

¹H-NMR (d₆-DMSO; 400 MHz): δ=9.92 (br. s, 1H); 9.14 (br. s, 1H); 7.75(br. s, 1 H); 7.38 (dd, 1H); 7.34 (dd, 1H); 7.28 (dd, 1H); 7.21 (dd,1H); 7.02 (dd, 1H); 6.96 (dd, 1H); 6.81 (dd, 1H); 6.71 (dd, 1H); 6.21(dd, 1H); 4.14 (s, 1H); 3.10 (q, 2H); 2.63 (m, 1H); 1.15 (t, 3H);0.87-0.81 (m, 4H).

LC-MS: retention time: 1.35 min

MS ES⁺: 548.0 [M+H]⁺

Example 10 Preparation ofN-(3-{3-[(4-ethynyl-2-fluorophenyl)amino]-5-fluoro-2-(sulfamoylamino)phenoxy}phenyl)ethanesulfonamide[Formic Acid salt]

Step 1

100 mgN-(3-{5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-(sulfamoylamino)-phenoxy}phenyl)ethanesulfonamide(0.16 mmol, 1 eq.), 3.7 mg Pd(dba)₂ (0.006 mmol; 4 mol %), 1.2 mg CuI(0.006 mmol; 4 mol %) and 8.4 mg PPh₃ (0.03 mmol; 20 mol %) are weighedinto a microwave vial, dissolved in 1.6 mL Et₃N, set under an Aratmosphere and supplied with 133 μL trimethylsilyl acetylene (0.961mmol, 6 eq.). The vial was capped and heated to 60° C. for 3 h uponwhich LCMS analysis showed complete turnover. The reaction mixture wasconcentrated in vacuo and the crude product (containing unreactedstarting material) was used without further purification.

LC-MS: retention time: 1.52 min

MS ES⁻: 593.4 [M−H]⁻

Step 2

The crude product from step 1 was dissolved in 2 mL THF and treated with0.24 mL TBAF solution (1.0 M in THF; 0.24 mmol, 1.55 eq.) and stirred atrt for 16 h. The reaction mixture was diluted with ethyl acetate, washedwith sat. sodium bicarbonate solution and subsequently with sat. sodiumchloride solution, dried and filtered. After concentration in vacuo theresidue was purified by preparative HPLC purification yielding 2.2 mg ofthe target compound (3% yield over 2 steps).

¹H-NMR (d₆-DMSO; 300 MHz): δ=8.61 (br. s, 1H); 7.82 (s, 1H); 7.10-7.40(m, 5 H); 6.99-7.05 (m, 4H); 6.83 (d, 1H); 6.62 (d, 1H); 6.07 (dd, 1H);4.15 (s, 1H); 3.10 (q, 2H); 1.16 (t, 3H).

LC-MS: retention time: 1.44 min

MS ES⁺: 545.8 [M+HCO₂H]⁺

Example 11 Preparation ofN-{4-fluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-[3-(sulfamoylamino)phenoxy]phenyl}cyclopropanesulfonamide

Solution A: 63 mg Chlorsutfonylisocyanate were dissolved in 0.11 mL DCM,heated to reflux and treated with 18 μL formic acid and 340 μL DMA.

125 mgN-{2-(3-aminophenoxy)-4-fluoro-6-[(2-fluoro-4-iodophenyl)amino]phenyl}-cyclopropanesulfonamide(0.224 mmol, 1 eq.) were dissolved in 0.76 mL DMA, and treated with 47μL DIPEA. Solution A was added by syringe and the resulting mixture wasstirred at rt for 16 h. The reaction mixture was portioned between waterand ethyl acetate, the aqueous layer reextracted with ethyl acetate, thecombined organic layers dried, filtered and concentrated in vacuo. Flashcolumn chromatography provided 77 mg of the target compound (34% yield).

¹H-NMR (d₆-DMSO; 400 MHz): δ=9.66 (br. s, 1H); 9.12 (br. s, 1H); 7.64(dd, 1H); 7.58 (s, 1H); 7.44 (d, 1H); 7.29 (t, 1H); 7.15 (s, 2H); 7.11(t, 1H); 6.99 (dd, 1H); 6.91 (t, 1H); 6.70 (dd, 1H); 6.56 (dd, 1H); 6.06(dd, 1H); 2.62-2.69 (m, 1H); 0.81-0.90 (m, 4H).

LC-MS: retention time: 1.36 min

MS ES⁻: 636.7 [M−H]⁻

Example 12 Preparation ofN-(4-fluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-{3-[(isopropylsulfonyl)amino]phenoxy}phenyl)cyclopropanesulfonamide

125 mgN-{2-(3-aminophenoxy)-4-fluoro-6-[(2-fluoro-4-iodophenyl)amino]phenyl}-cyclopropanesulfonamide(0.22 mmol, 1 eq.) were dissolved in 1 mL pyridine, and treated with 31μL isopropyl sulfonyl chloride (0.27 mmol, 1.2 eq.) and stirred at rtfor 16 h. The reaction mixture was quenched with water, diluted withtoluene and concentrated in vacuo. The residue was portioned betweenwater and ethyl acetate, the aqueous layer reextracted with ethylacetate, the combined organic layers dried, filtered and concentrated invacuo. Preparative HPLC purification followed by flash columnchromatography provided 70 mg of the target compound (47% yield).

¹H-NMR (d₆-DMSO; 400 MHz): δ=9.89 (br. s, 1H); 9.10 (br. s, 1H); 7.64(dd, 1H); 7.59 (br. s, 1H); 7.44 (d, 1H); 7.32 (t, 1H); 7.12 (t, 1H);7.03 (dd, 1H); 6.97 (t, 1 H); 6.79 (dd, 1H); 6.57 (dd, 1H); 6.11 (dd,1H); 3.21-3.28 (m, 1H); 2.61-2.67 (m, 1H); 1.20 (d, 6H); 0.80-0.88 (m,4H).

LC-MS: retention time: 1.49 min

MS ES⁺: 663.8 [M+H]⁺

The following example compound 13 was prepared in analogy to examplecompound 12 by treatingN-{2-(3-aminophenoxy)-4-fluoro-6-[(2-fluoro-4-iodophenyl)amino]phenyl}cyclopropanesulfonamide(Intermediate 11.A) with the respective commercially available sulfonylchlorides in the presence of pyridine.

Example Structure Name Analytical Data 13

N-(4-fluoro-2-[(2- fluoro-4- iodophenyl)amino]- 6-{3-[(methylsulfonyl)amino] phenoxy}phenyl) cyclopropanesulfonamide ¹H-NMR(d₆-DMSO; 300 MHz): δ = 9.83 (br. s, 1 H); 9.16 (br. s, 1 H); 7.64 (dd,1 H); 7.59 (s, 1 H); 7.45 (br. d, 1 H); 7.34 (dd, 1 H); 7.12 (dd, 1 H);7.01 (br. d, 1 H); 6.93 (dd, 1 H); 6.82 (dd, 1 H); 6.57 (dd, 1 H); 6.16(dd, 1 H); 2.99 (s, 3 H); 2.65 (m, 1 H); 0.88 - 0.78 (m, 4 H). LC-MS:retention time: 1.42 min MS ES⁺: 635.8 [M + H]⁺

Example 14 Preparation ofN-{4-fluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-[4-fluoro-3-(sulfamoylamino)phenoxy]phenyl}cyclopropanesulfonamide

547 mg crudeN-(5-{2-amino-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}-2-fluorophenyl)sulfuricdiamide (Intermediate 14.A) (0.993 mmol, 1. eq.) was dissolved in 8.5 mLpyridine, cooled to 0° C. and treated with 140 mg cyclopropanesulfonylchloride (0.993 mmol, 1 eq.). The reaction mixture was stirred at rt for18 h. Another 70 mg of cyclopropanesulfonyl chloride (0.497 mmol, 0.5eq.) were added and stirring was continued for 24 h. The reactionmixture diluted with toluene and concentrated in vacuo. The residue waspartitioned between water and ethyl acetate and the aqueouse layerreextracted twice with ethyl acetate. The combined organic layers werewashed once with half saturated brine and once with brine, filtered andconcentrated in vacuo. The crude product was purified by flash columnchromatography (n-hexane/ethyl acetate gradient) providing 40 mg of thedesired product (6% yield).

¹H-NMR (d₆-DMSO; 400 MHz): δ=9.38 (br. s, 1H); 9.11 (br. s, 1H); 7.64(dd, 1H); 7.58 (br. s, 1H); 7.44 (br. d, 1H); 7.28-7.22 (m, 2H); 7.17(d, 2H); 7.11 (dd, 1H); 6.83 (dm, 1H); 6.55 (dd, 1H); 6.07 (dd, 1H);2.71 (m, 1H); 0.89-0.82 (m, 4H).

LC-MS: retention time: 1.37 min

MS ES⁺: 655.34 [M+H]⁺

Example 15 Preparation ofN-(5-{2-[(cyclopropylsulfonyl)amino]-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}-2-fluorophenyl)cyclopropanesulfonamide

26.06 mg (0.04 mmol, 4% yield) ofN-(5-{2-[(cyclopropylsulfonyl)amino]-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}-2-fluorophenyl)cyclopropanesulfonamidewere isolated as a side product from the reaction mixture for examplecompound 14.

¹H-NMR (d₆-DMSO; 300 MHz): δ=9.79 (br. s, 1H); 9.09 (br. s, 1H); 7.65(dd, 1H); 7.60 (br. s, 1H); 7.45 (br. d, 1H); 7.31 (dd, 1H); 7.17-7.09(m, 2H); 6.98 (m, 1H); 6.55 (dd, 1H); 6.08 (dd, 1H); 2.73-2.61 (m, 2H);0.95-079 (m, 8H).

LC-MS: retention time: 1.44 min

MS ES⁺: 679.7 [M+H]⁺

Example 16 Preparation ofN-(2-fluoro-5-{5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-(sulfamoylamino)phenoxy}phenyl)sulfuricdiamide

2.36 mg (3.64 μmol, 0.37% yield) ofN-(2-fluoro-5-{5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-(sulfamoylamino)phenoxy}phenyl)sulfuricdiamide were isolated as a side product from the reaction mixture forexample compound 14.

¹H-NMR (d₆-DMSO; 300 MHz): δ=9.35 (br. s, 1H); 8.59 (br. s, 1H); 7.64(dd, 1H); 7.64 (br. s, 1H); 7.29-7.10 (m, 7H); 6.99 (br. s, 1H); 6.83(ddd, 1H); 6.47 (dd, 1 H); 5.99 (dd, 1H).

LC-MS: retention time: 1.24 min

MS ES⁺: 630.3 [M+H]⁺

Example 17 Preparation ofN-(4-fluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-{3-[(sulfamoylamino)methyl]phenoxy}phenyl)cyclopropanesulfonamide

814 mg of tert-butyl[(3-{2-[(cyclopropylsulfonyl)amino]-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}benzyl)sulfamoyl]carbamate(Intermediate 18A; 1.08 mmol, 1 eq.) were dissolved in 30 mL DCM and1.67 mL TFA were added. The reaction mixture was stirred at rtovernight. The reaction mixture was partitioned between saturated aq.sodium bicarbonate solution and dichloromethane. The pH was adjusted topH=8 with aq. 2N sodium hydroxide solution. The layers were separatedand the aqueouse layer reextracted twice with dichloromethane. Thecombined organic layers were washed once with brine, filtered andconcentrated in vacuo. The crude product was purified by preparativeHPLC providing 322 mg of the desired product (46% yield).

¹H-NMR (d₆-DMSO; 400 MHz): δ=9.11 (br. s, 1H); 7.64 (dd, 1H); 7.58 (br.s, 1H); 7.44 (br. d, 1H); 7.34 (dd, 1H); 7.18 (d, 1H); 7.15-7.08 (m,3H); 6.99 (br. d, 1H); 6.62 (s, 2H); 6.55 (dd, 1H); 6.02 (dd, 1H); 4.07(d, 2H); 2.68 (m, 1H); 0.89-0.82 (m, 4H).

LC-MS: retention time: 1.37 min

MS ES⁺: 650.8 [M+H]⁺

Further, the compounds of formula (I) of the present invention can beconverted to any salt as described herein, by any method which is knownto the person skilled in the art. Similarly, any salt of a compound offormula (1) of the present invention can be converted into the freecompound, by any method which is known to the person skilled in the art.

Pharmaceutical Compositions of the Compounds of the Invention

This invention also relates to pharmaceutical compositions containingone or more compounds of the present invention. These compositions canbe utilised to achieve the desired pharmacological effect byadministration to a patient in need thereof. A patient, for the purposeof this invention, is a mammal, including a human, in need of treatmentfor the particular condition or disease. Therefore, the presentinvention includes pharmaceutical compositions that are comprised of apharmaceutically acceptable carrier and a pharmaceutically effectiveamount of a compound, or salt thereof, of the present invention. Apharmaceutically acceptable carrier is preferably a carrier that isrelatively non-toxic and innocuous to a patient at concentrationsconsistent with effective activity of the active ingredient so that anyside effects ascribable to the carrier do not vitiate the beneficialeffects of the active ingredient. A pharmaceutically effective amount ofcompound is preferably that amount which produces a result or exerts aninfluence on the particular condition being treated. The compounds ofthe present invention can be administered withpharmaceutically-acceptable carriers well known in the art using anyeffective conventional dosage unit forms, including immediate, slow andtimed release preparations, orally, parenterally, topically, nasally,ophthalmically, optically, sublingually, rectally, vaginally, and thelike.

For oral administration, the compounds can be formulated into solid orliquid preparations such as capsules, pills, tablets, troches, lozenges,melts, powders, solutions, suspensions, or emulsions, and may beprepared according to methods known to the art for the manufacture ofpharmaceutical compositions. The solid unit dosage forms can be acapsule that can be of the ordinary hard- or soft-shelled gelatin typecontaining, for example, surfactants, lubricants, and inert fillers suchas lactose, sucrose, calcium phosphate, and corn starch.

In another embodiment, the compounds of this invention may be tabletedwith conventional tablet bases such as lactose, sucrose and cornstarchin combination with binders such as acacia, corn starch or gelatin,disintegrating agents intended to assist the break-up and dissolution ofthe tablet following administration such as potato starch, alginic acid,corn starch, and guar gum, gum tragacanth, acacia, lubricants intendedto improve the flow of tablet granulation and to prevent the adhesion oftablet material to the surfaces of the tablet dies and punches, forexample talc, stearic acid, or magnesium, calcium or zinc stearate,dyes, coloring agents, and flavoring agents such as peppermint, oil ofwintergreen, or cherry flavoring, intended to enhance the aestheticqualities of the tablets and make them more acceptable to the patient.Suitable excipients for use in oral liquid dosage forms includedicalcium phosphate and diluents such as water and alcohols, forexample, ethanol, benzyl alcohol, and polyethylene alcohols, either withor without the addition of a pharmaceutically acceptable surfactant,suspending agent or emulsifying agent. Various other materials may bepresent as coatings or to otherwise modify the physical form of thedosage unit. For instance tablets, pills or capsules may be coated withshellac, sugar or both.

Dispersible powders and granules are suitable for the preparation of anaqueous suspension. They provide the active ingredient in admixture witha dispersing or wetting agent, a suspending agent and one or morepreservatives. Suitable dispersing or wetting agents and suspendingagents are exemplified by those already mentioned above. Additionalexcipients, for example those sweetening, flavoring and coloring agentsdescribed above, may also be present.

The pharmaceutical compositions of this invention may also be in theform of oil-in-water emulsions. The oily phase may be a vegetable oilsuch as liquid paraffin or a mixture of vegetable oils. Suitableemulsifying agents may be (1) naturally occurring gums such as gumacacia and gum tragacanth, (2) naturally occurring phosphatides such assoy bean and lecithin, (3) esters or partial esters derived form fattyacids and hexitol anhydrides, for example, sorbitan monooleate, (4)condensation products of said partial esters with ethylene oxide, forexample, polyoxyethylene sorbitan monooleate. The emulsions may alsocontain sweetening and flavoring agents.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil such as, for example, arachis oil, olive oil, sesameoil or coconut oil, or in a mineral oil such as liquid paraffin. Theoily suspensions may contain a thickening agent such as, for example,beeswax, hard paraffin, or cetyl alcohol. The suspensions may alsocontain one or more preservatives, for example, ethyl or n-propylp-hydroxybenzoate; one or more coloring agents; one or more flavoringagents; and one or more sweetening agents such as sucrose or saccharin.

Syrups and elixirs may be formulated with sweetening agents such as, forexample, glycerol, propylene glycol, sorbitol or sucrose. Suchformulations may also contain a demulcent, and preservative, such asmethyl and propyl parabens and flavoring and coloring agents.

The compounds of this invention may also be administered parenterally,that is, subcutaneously, intravenously, intraocularly, intrasynovially,intramuscularly, or interperitoneally, as injectable dosages of thecompound in preferably a physiologically acceptable diluent with apharmaceutical carrier which can be a sterile liquid or mixture ofliquids such as water, saline, aqueous dextrose and related sugarsolutions, an alcohol such as ethanol, isopropanol, or hexadecylalcohol, glycols such as propylene glycol or polyethylene glycol,glycerol ketals such as 2,2-dimethyl-1,1-dioxolane-4-methanol, etherssuch as poly(ethylene glycol) 400, an oil, a fatty acid, a fatty acidester or, a fatty acid glyceride, or an acetylated fatty acid glyceride,with or without the addition of a pharmaceutically acceptable surfactantsuch as a soap or a detergent, suspending agent such as pectin,carbomers, methycellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agent and other pharmaceuticaladjuvants.

Illustrative of oils which can be used in the parenteral formulations ofthis invention are those of petroleum, animal, vegetable, or syntheticorigin, for example, peanut oil, soybean oil, sesame oil, cottonseedoil, corn oil, olive oil, petrolatum and mineral oil. Suitable fattyacids include oleic acid, stearic acid, isostearic acid and myristicacid. Suitable fatty acid esters are, for example, ethyl oleate andisopropyl myristate. Suitable soaps include fatty acid alkali metal,ammonium, and triethanolamine salts and suitable detergents includecationic detergents, for example dimethyl dialkyl ammonium halides,alkyl pyridinium halides, and alkylamine acetates; anionic detergents,for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether,and monoglyceride sulfates, and sulfosuccinates; non-ionic detergents,for example, fatty amine oxides, fatty acid alkanolamides, andpolyoxyethylene-oxypropylene)s or ethylene oxide or propylene oxidecopolymers; and amphoteric detergents, for example,alkyl-beta-aminopropionates, and 2-alkylimidazoline quarternary ammoniumsalts, as well as mixtures.

The parenteral compositions of this invention will typically containfrom about 0.5% to about 25% by weight of the active ingredient insolution. Preservatives and buffers may also be used advantageously. Inorder to minimise or eliminate irritation at the site of injection, suchcompositions may contain a non-ionic surfactant having ahydrophile-lipophile balance (HLB) preferably of from about 12 to about17. The quantity of surfactant in such formulation preferably rangesfrom about 5% to about 15% by weight. The surfactant can be a singlecomponent having the above HLB or can be a mixture of two or morecomponents having the desired HLB.

Illustrative of surfactants used in parenteral formulations are theclass of polyethylene sorbitan fatty acid esters, for example, sorbitanmonooleate and the high molecular weight adducts of ethylene oxide witha hydrophobic base, formed by the condensation of propylene oxide withpropylene glycol.

The pharmaceutical compositions may be in the form of sterile injectableaqueous suspensions. Such suspensions may be formulated according toknown methods using suitable dispersing or wetting agents and suspendingagents such as, for example, sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents which may be a naturally occurring phosphatide such aslecithin, a condensation product of an alkylene oxide with a fatty acid,for example, polyoxyethylene stearate, a condensation product ofethylene oxide with a long chain aliphatic alcohol, for example,heptadeca-ethyleneoxycetanol, a condensation product of ethylene oxidewith a partial ester derived form a fatty acid and a hexitol such aspolyoxyethylene sorbitol monooleate, or a condensation product of anethylene oxide with a partial ester derived from a fatty acid and ahexitol anhydride, for example polyoxyethylene sorbitan monooleate.

The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent. Diluents and solvents that may be employed are, for example,water, Ringer's solution, isotonic sodium chloride solutions andisotonic glucose solutions. In addition, sterile fixed oils areconventionally employed as solvents or suspending media. For thispurpose, any bland, fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid can be usedin the preparation of injectables.

A composition of the invention may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritationexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials are, for example, cocoa butter and polyethyleneglycol.

Another formulation employed in the methods of the present inventionemploys transdermal delivery devices (“patches”). Such transdermalpatches may be used to provide continuous or discontinuous infusion ofthe compounds of the present invention in controlled amounts. Theconstruction and use of transdermal patches for the delivery ofpharmaceutical agents is well known in the art (see, e.g., U.S. Pat. No.5,023,252, issued Jun. 11, 1991, incorporated herein by reference). Suchpatches may be constructed for continuous, pulsatile, or on demanddelivery of pharmaceutical agents.

Controlled release formulations for parenteral administration includeliposomal, polymeric microsphere and polymeric gel formulations that areknown in the art.

It may be desirable or necessary to introduce the pharmaceuticalcomposition to the patient via a mechanical delivery device. Theconstruction and use of mechanical delivery devices for the delivery ofpharmaceutical agents is well known in the art. Direct techniques for,for example, administering a drug directly to the brain usually involveplacement of a drug delivery catheter into the patient's ventricularsystem to bypass the blood-brain barrier. One such implantable deliverysystem, used for the transport of agents to specific anatomical regionsof the body, is described in U.S. Pat. No. 5,011,472, issued Apr. 30,1991.

The compositions of the invention can also contain other conventionalpharmaceutically acceptable compounding ingredients, generally referredto as carriers or diluents, as necessary or desired. Conventionalprocedures for preparing such compositions in appropriate dosage formscan be utilized. Such ingredients and procedures include those describedin the following references, each of which is incorporated herein byreference: Powell, M. F. et al., “Compendium of Excipients forParenteral Formulations” PDA Journal of Pharmaceutical Science aTechnology 1998, 52(5), 238-311; Strickley, R. G “ParenteralFormulations of Small Molecule Therapeutics Marketed in the UnitedStates (1999)-Part-1” PDA Journal of Pharmaceutical Science & Technology1999, 53(6), 324-349; and Nema, S. et al., “Excipients and Their Use inInjectable Products” PDA Journal of Pharmaceutical Science & Technology1997, 51(4), 166-171.

Commonly used pharmaceutical ingredients that can be used as appropriateto formulate the composition for its intended route of administrationinclude:

acidifying agents (examples include but are not limited to acetic acid,citric acid, fumaric acid, hydrochloric acid, nitric acid);

alkalinizing agents (examples include but are not limited to ammoniasolution, ammonium carbonate, diethanolamine, monoethanolamine,potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide,triethanolamine, trolamine);

adsorbents (examples include but are not limited to powdered celluloseand activated charcoal);

aerosol propellants (examples include but are not limited to carbondioxide, CCl₂F₂, F₂ClC—CClF₂ and CClF₃)

air displacement agents (examples include but are not limited tonitrogen and argon);

antifungal preservatives (examples include but are not limited tobenzoic acid, butylparaben, ethylparaben, methylparaben, propylparaben,sodium benzoate);

antimicrobial preservatives (examples include but are not limited tobenzalkonium chloride, benzethonium chloride, benzyl alcohol,cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol,phenylmercuric nitrate and thimerosal);

antioxidants (examples include but are not limited to ascorbic acid,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorus acid, monothioglycerol, propyl gallate, sodium ascorbate,sodium bisulfite, sodium formaldehyde sulfoxylate, sodiummetabisulfite);

binding materials (examples include but are not limited to blockpolymers, natural and synthetic rubber, polyacrylates, polyurethanes,silicones, polysiloxanes and styrene-butadiene copolymers);

buffering agents (examples include but are not limited to potassiummetaphosphate, dipotassium phosphate, sodium acetate, sodium citrateanhydrous and sodium citrate dihydrate)

carrying agents (examples include but are not limited to acacia syrup,aromatic syrup, aromatic elixir, cherry syrup, cocoa syrup, orangesyrup, syrup, corn oil, mineral oil, peanut oil, sesame oil,bacteriostatic sodium chloride injection and bacteriostatic water forinjection)

chelating agents (examples include but are not limited to edetatedisodium and edetic acid)

colorants (examples include but are not limited to FD&C Red No. 3, FD&CRed No. 20, FD&C Yellow No. 6, FD&C Blue No. 2, D&C Green No. 5, D&COrange No. 5, D&C Red No. 8, caramel and ferric oxide red);

clarifying agents (examples include but are not limited to bentonite);

emulsifying agents (examples include but are not limited to acacia,cetomacrogol, cetyl alcohol, glyceryl monostearate, lecithin, sorbitanmonooleate, polyoxyethylene 50 monostearate);

encapsulating agents (examples include but are not limited to gelatinand cellulose acetate phthalate)

flavorants (examples include but are not limited to anise oil, cinnamonoil, cocoa, menthol, orange oil, peppermint oil and vanillin);

humectants (examples include but are not limited to glycerol, propyleneglycol and sorbitol);

levigating agents (examples include but are not limited to mineral oiland glycerin);

oils (examples include but are not limited to arachis oil, mineral oil,olive oil, peanut oil, sesame oil and vegetable oil);

ointment bases (examples include but are not limited to lanolin,hydrophilic ointment, polyethylene glycol ointment, petrolatum,hydrophilic petrolatum, white ointment, yellow ointment, and rose waterointment);

penetration enhancers (transdermal delivery) (examples include but arenot limited to monohydroxy or polyhydroxy alcohols, mono- or polyvalentalcohols, saturated or unsaturated fatty alcohols, saturated orunsaturated fatty esters, saturated or unsaturated dicarboxylic acids,essential oils, phosphatidyl derivatives, cephalin, terpenes, amides,ethers, ketones and ureas)

plasticizers (examples include but are not limited to diethyl phthalateand glycerol);

solvents (examples include but are not limited to ethanol, corn oil,cottonseed oil, glycerol, isopropanol, mineral oil, oleic acid, peanutoil, purified water, water for injection, sterile water for injectionand sterile water for irrigation);

stiffening agents (examples include but are not limited to cetylalcohol, cetyl esters wax, microcrystalline wax, paraffin, stearylalcohol, white wax and yellow wax);

suppository bases (examples include but are not limited to cocoa butterand polyethylene glycols (mixtures));

surfactants (examples include but are not limited to benzalkoniumchloride, nonoxynol 10, oxtoxynol 9, polysorbate 80, sodium laurylsulfate and sorbitan mono-palmitate);

suspending agents (examples include but are not limited to agar,bentonite, carbomers, carboxymethylcellulose sodium, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,kaolin, methylcellulose, tragacanth and veegum);

sweetening agents (examples include but are not limited to aspartame,dextrose, glycerol, mannitol, propylene glycol, saccharin sodium,sorbitol and sucrose);

tablet anti-adherents (examples include but are not limited to magnesiumstearate and talc);

tablet binders (examples include but are not limited to acacia, alginicacid, carboxymethylcellulose sodium, compressible sugar, ethylcellulose,gelatin, liquid glucose, methylcellulose, non-crosslinked polyvinylpyrrolidone, and pregelatinized starch);

tablet and capsule diluents (examples include but are not limited todibasic calcium phosphate, kaolin, lactose, mannitol, microcrystallinecellulose, powdered cellulose, precipitated calcium carbonate, sodiumcarbonate, sodium phosphate, sorbitol and starch);

tablet coating agents (examples include but are not limited to liquidglucose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, methylcellulose, ethylcellulose, cellulose acetatephthalate and shellac);

tablet direct compression excipients (examples include but are notlimited to dibasic calcium phosphate);

tablet disintegrants (examples include but are not limited to alginicacid, carboxymethylcellulose calcium, microcrystalline cellulose,polacrillin potassium, cross-linked polyvinylpyrrolidone, sodiumalginate, sodium starch glycollate and starch);

tablet glidants (examples include but are not limited to colloidalsilica, corn starch and talc);

tablet lubricants (examples include but are not limited to calciumstearate, magnesium stearate, mineral oil, stearic acid and zincstearate);

tablet/capsule opaquants (examples include but are not limited totitanium dioxide);

tablet polishing agents (examples include but are not limited to carnubawax and white wax);

thickening agents (examples include but are not limited to beeswax,cetyl alcohol and paraffin);

tonicity agents (examples include but are not limited to dextrose andsodium chloride);

viscosity increasing agents (examples include but are not limited toalginic acid, bentonite, carbomers, carboxymethylcellulose sodium,methylcellulose, polyvinyl pyrrolidone, sodium alginate and tragacanth);and

wetting agents (examples include but are not limited toheptadecaethylene oxycetanol, lecithins, sorbitol monooleate,polyoxyethylene sorbitol monooleate, and polyoxyethylene stearate).

Pharmaceutical compositions according to the present invention can beillustrated as follows:

Sterile IV Solution: A 5 mg/mL solution of the desired compound of thisinvention can be made using sterile, injectable water, and the pH isadjusted if necessary. The solution is diluted for administration to 1-2mg/mL with sterile 5% dextrose and is administered as an IV infusionover about 60 minutes.

Lyophilised powder for IV administration: A sterile preparation can beprepared with (i) 100-1000 mg of the desired compound of this inventionas a lyophilised powder, (ii) 32-327 mg/mL sodium citrate, and (iii)300-3000 mg Dextran 40. The formulation is reconstituted with sterile,injectable saline or dextrose 5% to a concentration of 10 to 20 mg/mL,which is further diluted with saline or dextrose 5% to 0.2-0.4 mg/mL,and is administered either IV bolus or by IV infusion over 15-60minutes.

Intramuscular suspension: The following solution or suspension can beprepared, for intramuscular injection:

50 mg/mL of the desired, water-insoluble compound of this invention5 mg/mL sodium carboxymethylcellulose4 mg/mL TWEEN 809 mg/mL sodium chloride9 mg/mL benzyl alcohol

Hard Shell Capsules: A large number of unit capsules are prepared byfilling standard two-piece hard galantine capsules each with 100 mg ofpowdered active ingredient, 150 mg of lactose, 50 mg of cellulose and 6mg of magnesium stearate.

Soft Gelatin Capsules: A mixture of active ingredient in a digestibleoil such as soybean oil, cottonseed oil or olive oil is prepared andinjected by means of a positive displacement pump into molten gelatin toform soft gelatin capsules containing 100 mg of the active ingredient.The capsules are washed and dried. The active ingredient can bedissolved in a mixture of polyethylene glycol, glycerin and sorbitol toprepare a water miscible medicine mix.

Tablets: A large number of tablets are prepared by conventionalprocedures so that the dosage unit is 100 mg of active ingredient, 0.2mg. of colloidal silicon dioxide, 5 mg of magnesium stearate, 275 mg ofmicrocrystalline cellulose, 11 mg. of starch, and 98.8 mg of lactose.Appropriate aqueous and non-aqueous coatings may be applied to increasepalatability, improve elegance and stability or delay absorption.

Immediate Release Tablets/Capsules: These are solid oral dosage formsmade by conventional and novel processes. These units are taken orallywithout water for immediate dissolution and delivery of the medication.The active ingredient is mixed in a liquid containing ingredient such assugar, gelatin, pectin and sweeteners. These liquids are solidified intosolid tablets or caplets by freeze drying and solid state extractiontechniques. The drug compounds may be compressed with viscoelastic andthermoelastic sugars and polymers or effervescent components to produceporous matrices intended for immediate release, without the need ofwater.

Combination Therapies

The compounds of this invention can be administered as the solepharmaceutical agent or in combination with one or more otherpharmaceutical agents where the combination causes no unacceptableadverse effects. The present invention relates also to suchcombinations. For example, the compounds of this invention can becombined with known anti-hyper-proliferative or other indication agents,and the like, as well as with admixtures and combinations thereof. Otherindication agents include, but are not limited to, anti-angiogenicagents, mitotic inhibitors, alkylating agents, anti-metabolites,DNA-intercalating antibiotics, growth factor inhibitors, cell cycleinhibitors, enzyme inhibitors, toposisomerase inhibitors, biologicalresponse modifiers, or anti-hormones.

The additional pharmaceutical agent can be aldesleukin, alendronic acid,alfaferone, alitretinoin, allopurinol, aloprim, aloxi, altretamine,aminoglutethimide, amifostine, amrubicin, amsacrine, anastrozole,anzmet, aranesp, arglabin, arsenic trioxide, aromasin, 5-azacytidine,azathioprine, BCG or tice BCG, bestatin, betamethasone acetate,betamethasone sodium phosphate, bexarotene, bleomycin sulfate,broxuridine, bortezomib, busulfan, calcitonin, campath, capecitabine,carboplatin, casodex, cefesone, celmoleukin, cerubidine, chlorambucil,cisplatin, cladribine, cladribine, clodronic acid, cyclophosphamide,cytarabine, dacarbazine, dactinomycin, DaunoXome, decadron, decadronphosphate, delestrogen, denileukin diftitox, depomedrol, deslorelin,dexrazoxane, diethylstilbestrol, diflucan, docetaxel, doxifluridine,doxorubicin, dronabinol, DW-166HC, eligard, elitek, ellence, emend,epirubicin, epoetin alfa, epogen, eptaplatin, ergamisol, estrace,estradiol, estramustine phosphate sodium, ethinyl estradiol, ethyol,etidronic acid, etopophos, etoposide, fadrozole, farston, filgrastim,finasteride, fligrastim, floxuridine, fluconazole, fludarabine,5-fluorodeoxyuridine monophosphate, 5-fluorouracil (5-FU),fluoxymesterone, flutamide, formestane, fosteabine, fotemustine,fulvestrant, gammagard, gemcitabine, gemtuzumab, gleevec, gliadel,goserelin, granisetron HCl, histrelin, hycamtin, hydrocortone,eyrthro-hydroxynonyladenine, hydroxyurea, ibritumomab tiuxetan,idarubicin, ifosfamide, interferon alpha, interferon-alpha 2, interferonalfa-2A, interferon alfa-2B, interferon alfa-n1, interferon alfa-n3,interferon beta, interferon gamma-1a, interleukin-2, intron A, iressa,irinotecan, kytril, lentinan sulfate, letrozole, leucovorin, leuprolide,leuprolide acetate, levamisole, levofolinic acid calcium salt,levothroid, levoxyl, lomustine, lonidamine, marinol, mechlorethamine,mecobalamin, medroxyprogesterone acetate, megestrol acetate, melphalan,menest, 6-mercaptopurine, Mesna, methotrexate, metvix, miltefosine,minocycline, mitomycin C, mitotane, mitoxantrone, Modrenal, Myocet,nedaplatin, neulasta, neumega, neupogen, nilutamide, nolvadex,NSC-631570, OCT-43, octreotide, ondansetron HCl, orapred, oxaliplatin,paclitaxel, pediapred, pegaspargase, Pegasys, pentostatin, picibanil,pilocarpine HCl, pirarubicin, plicamycin, porfimer sodium,prednimustine, prednisolone, prednisone, premarin, procarbazine,procrit, raltitrexed, rebif, rhenium-186 etidronate, rituximab,roferon-A, romurtide, salagen, sandostatin, sargramostim, semustine,sizofuran, sobuzoxane, solu-medrol, sparfosic acid, stem-cell therapy,streptozocin, strontium-89 chloride, synthroid, tamoxifen, tamsulosin,tasonermin, tastolactone, taxotere, teceleukin, temozolomide,teniposide, testosterone propionate, testred, thioguanine, thiotepa,thyrotropin, tiludronic acid, topotecan, toremifene, tositumomab,trastuzumab, treosulfan, tretinoin, trexall, trimethylmelamine,trimetrexate, triptorelin acetate, triptorelin pamoate, UFT, uridine,valrubicin, vesnarinone, vinblastine, vincristine, vindesine,vinorelbine, virulizin, zinecard, zinostatin stimalamer, zofran,ABI-007, acolbifene, actimmune, affinitak, aminopterin, arzoxifene,asoprisnil, atamestane, atrasentan, sorafenib, avastin, CCI-779,CDC-501, celebrex, cetuximab, crisnatol, cyproterone acetate,decitabine, DN101, doxorubicin-MTC, dSLIM, dutasteride, edotecarin,eflornithine, exatecan, fenretinide, histamine dihydrochloride,histrelin hydrogel implant, holmium-166 DOTMP, ibandronic acid,interferon gamma, intron-PEG, ixabepilone, keyhole limpet hemocyanin,L-651582, lanreotide, lasofoxifene, Libra, lonafarnib, miproxifene,minodronate, MS-209, liposomal MTP-PE, MX-6, nafarelin, nemorubicin,neovastat, nolatrexed, oblimersen, onco-TCS, osidem, paclitaxelpolyglutamate, pamidronate disodium, PN-401, QS-21, quazepam, R-1549,raloxifene, ranpirnase, 13-cis-retinoic acid, satraplatin, seocalcitol,T-138067, tarceva, taxoprexin, thymosin alpha 1, tiazofurine,tipifarnib, tirapazamine, TLK-286, toremifene, TransMID-107R, valspodar,vapreotide, vatalanib, verteporfin, vinflunine, Z-100, zoledronic acidor combinations thereof.

Optional anti-hyper-proliferative agents which can be added to thecomposition include but are not limited to compounds listed on thecancer chemotherapy drug regimens in the 11^(th) Edition of the MerckIndex, (1996), which is hereby incorporated by reference, such asasparaginase, bleomycin, carboplatin, carmustine, chlorambucil,cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine,dactinomycin, daunorubicin, doxorubicin (adriamycine), epirubicin,etoposide, 5-fluorouracil, hexamethylmelamine, hydroxyurea, ifosfamide,irinotecan, leucovorin, lomustine, mechlorethamine, 6-mercaptopurine,mesna, methotrexate, mitomycin C, mitoxantrone, prednisolone,prednisone, procarbazine, raloxifen, streptozocin, tamoxifen,thioguanine, topotecan, vinblastine, vincristine, and vindesine.

Other anti-hyper-proliferative agents suitable for use with thecomposition of the invention include but are not limited to thosecompounds acknowledged to be used in the treatment of neoplasticdiseases in Goodman and Gilman's The Pharmacological Basis ofTherapeutics (Ninth Edition), editor Molinoff et al., publ. byMcGraw-Hill, pages 1225-1287, (1996), which is hereby incorporated byreference, such as aminoglutethimide, L-asparaginase, azathioprine,5-azacytidine cladribine, busulfan, diethylstilbestrol,2′,2′-difluorodeoxycytidine, docetaxel, erythrohydroxynonyl adenine,ethinyl estradiol, 5-fluorodeoxyuridine, 5-fluorodeoxyuridinemono-phosphate, fludarabine phosphate, fluoxymesterone, flutamide,hydroxyprogesterone caproate, idarubicin, interferon,medroxyprogesterone acetate, megestrol acetate, melphalan, mitotane,paclitaxel, pentostatin, N-phosphonoacetyl-L-aspartate (PALA),plicamycin, semustine, teniposide, testosterone propionate, thiotepa,trimethyl-melamine, uridine, and vinorelbine.

Other anti-hyper-proliferative agents suitable for use with thecomposition of the invention include but are not limited to otheranti-cancer agents such as epothilone and its derivatives, irinotecan,raloxifen and topotecan.

The compounds of the invention may also be administered in combinationwith protein therapeutics. Such protein therapeutics suitable for thetreatment of cancer or other angiogenic disorders and for use with thecompositions of the invention include, but are not limited to, aninterferon (e.g., interferon .alpha., .beta., or .gamma.) supraagonisticmonoclonal antibodies, Tuebingen, TRP-1 protein vaccine, Colostrinin,anti-FAP antibody, YH-16, gemtuzumab, infliximab, cetuximab,trastuzumab, denileukin diftitox, rituximab, thymosin alpha 1,bevacizumab, mecasermin, mecasermin rinfabate, oprelvekin, natalizumab,rhMBL, MFE-CP1+ZD-2767-P, ABT-828, ErbB2-specific immunotoxin, SGN-35,MT-103, rinfabate, AS-1402, B43-genistein, L-19 basedradioimmunotherapeutics, AC-9301, NY-ESO-1 vaccine, IMC-1C11, CT-322,rhCC10, r(m)CRP, MORAb-009, aviscumine, MDX-1307, Her-2 vaccine,APC-8024, NGR-hTNF, rhH1.3, IGN-311, Endostatin, volociximab, PRO-1762,lexatumumab, SGN-40, pertuzumab, EMD-273063, L19-IL-2 fusion protein,PRX-321, CNTO-328, MDX-214, tigapotide, CAT-3888, labetuzumab,alpha-particle-emitting radioisotope-llinked lintuzumab, EM-1421,HyperAcute vaccine, tucotuzumab celmoleukin, galiximab, HPV-16-E7,Javelin-prostate cancer, Javelin-melanoma, NY-ESO-1 vaccine, EGFvaccine, CYT-004-MelQbG10, WT1 peptide, oregovomab, ofatumumab,zatutumumab, cintredekin besudotox, WX-G250, Albuferon, aflibercept,denosumab, vaccine, CTP-37, efungumab, or 131I-chTNT-1/B. Monoclonalantibodies useful as the protein therapeutic include, but are notlimited to, muromonab-CD3, abciximab, edrecolomab, daclizumab,gentuzumab, alemtuzumab, ibritumomab, cetuximab, bevicizumab,efalizumab, adalimumab, omalizumab, muromomab-CD3, rituximab,daclizumab, trastuzumab, palivizumab, basiliximab, and infliximab.

Generally, the use of cytotoxic and/or cytostatic agents in combinationwith a compound or composition of the present invention will serve to:

-   (1) yield better efficacy in reducing the growth of a tumor or even    eliminate the tumor as compared to administration of either agent    alone,-   (2) provide for the administration of lesser amounts of the    administered chemo-therapeutic agents,-   (3) provide for a chemotherapeutic treatment that is well tolerated    in the patient with fewer deleterious pharmacological complications    than observed with single agent chemotherapies and certain other    combined therapies,-   (4) provide for treating a broader spectrum of different cancer    types in mammals, especially humans,-   (5) provide for a higher response rate among treated patients,-   (6) provide for a longer survival time among treated patients    compared to standard chemotherapy treatments,-   (7) provide a longer time for tumor progression, and/or-   (8) yield efficacy and tolerability results at least as good as    those of the agents used alone, compared to known instances where    other cancer agent combinations produce antagonistic effects.

Methods of Sensitizing Cells to Radiation

In a distinct embodiment of the present invention, a compound of thepresent invention may be used to sensitize a cell to radiation. That is,treatment of a cell with a compound of the present invention prior toradiation treatment of the cell renders the cell more susceptible to DNAdamage and cell death than the cell would be in the absence of anytreatment with a compound of the invention. In one aspect, the cell istreated with at least one compound of the invention.

Thus, the present invention also provides a method of killing a cell,wherein a cell is administered one or more compounds of the invention incombination with conventional radiation therapy.

The present invention also provides a method of rendering a cell moresusceptible to cell death, wherein the cell is treated one or morecompounds of the invention prior to the treatment of the cell to causeor induce cell death. In one aspect, after the cell is treated with oneor more compounds of the invention, the cell is treated with at leastone compound, or at least one method, or a combination thereof, in orderto cause DNA damage for the purpose of inhibiting the function of thenormal cell or killing the cell.

In one embodiment, a cell is killed by treating the cell with at leastone DNA damaging agent. That is, after treating a cell with one or morecompounds of the invention to sensitize the cell to cell death, the cellis treated with at least one DNA damaging agent to kill the cell. DNAdamaging agents useful in the present invention include, but are notlimited to, chemotherapeutic agents (e.g., cisplatinum), ionizingradiation (X-rays, ultraviolet radiation), carcinogenic agents, andmutagenic agents.

In another embodiment, a cell is killed by treating the cell with atleast one method to cause or induce DNA damage. Such methods include,but are not limited to, activation of a cell signalling pathway thatresults in DNA damage when the pathway is activated, inhibiting of acell signalling pathway that results in DNA damage when the pathway isinhibited, and inducing a biochemical change in a cell, wherein thechange results in DNA damage. By way of a non-limiting example, a DNArepair pathway in a cell can be inhibited, thereby preventing the repairof DNA damage and resulting in an abnormal accumulation of DNA damage ina cell.

In one aspect of the invention, a compound of the invention isadministered to a cell prior to the radiation or orther induction of DNAdamage in the cell. In another aspect of the invention, a compound ofthe invention is administered to a cell concomitantly with the radiationor orther induction of DNA damage in the cell. In yet another aspect ofthe invention, a compound of the invention is administered to a cellimmediately after radiation or orther induction of DNA damage in thecell has begun.

In another aspect, the cell is in vitro. In another embodiment, the cellis in vivo.

As mentioned supra, the compounds of the present invention havesurprisingly been found to effectively inhibit allo-MEK and maytherefore be used for the treatment or prophylaxis of diseases ofuncontrolled cell growth, proliferation and/or survival, inappropriatecellular immune responses, or inappropriate cellular inflammatoryresponses, or diseases which are accompanied with uncontrolled cellgrowth, proliferation and/or survival, inappropriate cellular immuneresponses, or inappropriate cellular inflammatory responses,particularly in which the uncontrolled cell growth, proliferation and/orsurvival, inappropriate cellular immune responses, or inappropriatecellular inflammatory responses is mediated by allo-MEK, such as, forexample, haematological tumours, solid tumours, and/or metastasesthereof, e.g. leukaemias and myelodysplastic syndrome, malignantlymphomas, head and neck tumours including brain tumours and brainmetastases, tumours of the thorax including non-small cell and smallcell lung tumours, gastrointestinal tumours, endocrine tumours, mammaryand other gynaecological tumours, urological tumours including renal,bladder and prostate tumours, skin tumours, and sarcomas, and/ormetastases thereof.

In accordance with another aspect therefore, the present inventioncovers a compound of general formula (I), or a stereoisomer, a tautomer,an N-oxide, a hydrate, a solvate, or a salt thereof, particularly apharmaceutically acceptable salt thereof, or a mixture of same, asdescribed and defined herein, for use in the treatment or prophylaxis ofa disease, as mentioned supra.

Another particular aspect of the present invention is therefore the useof a compound of general formula (I) described supra for manufacturing apharmaceutical composition for the treatment or prophylaxis of adisease.

The diseases referred to in the two preceding paragraphs are diseases ofuncontrolled cell growth, proliferation and/or survival, inappropriatecellular immune responses, or inappropriate cellular inflammatoryresponses, or diseases which are accompanied with uncontrolled cellgrowth, proliferation and/or survival, inappropriate cellular immuneresponses, or inappropriate cellular inflammatory responses,particularly in which the uncontrolled cell growth, proliferation and/orsurvival, inappropriate cellular immune responses, or inappropriatecellular inflammatory responses is mediated by Mps-1, such as, forexample, haematological tumours, solid tumours, and/or metastasesthereof, e.g. leukaemias and myelodysplastic syndrome, malignantlymphomas, head and neck tumours including brain tumours and brainmetastases, tumours of the thorax including non-small cell and smallcell lung tumours, gastrointestinal tumours, endocrine tumours, mammaryand other gynaecological tumours, urological tumours including renal,bladder and prostate tumours, skin tumours, and sarcomas, and/ormetastases thereof.

The term “inappropriate” within the context of the present invention, inparticular in the context of “inappropriate cellular immune responses,or inappropriate cellular inflammatory responses”, as used herein, is tobe understood as preferably meaning a response which is less than, orgreater than normal, and which is associated with, responsible for, orresults in, the pathology of said diseases.

Preferably, the use is in the treatment or prophylaxis of diseases,wherein the diseases are haemotological tumours, solid tumours and/ormetastases thereof.

Method of Treating Hyper-Proliferative Disorders

The present invention relates to a method for using the compounds of thepresent invention and compositions thereof, to treat mammalianhyper-proliferative disorders. Compounds can be utilized to inhibit,block, reduce, decrease, etc., cell proliferation and/or cell division,and/or produce apoptosis. This method comprises administering to amammal in need thereof, including a human, an amount of a compound ofthis invention, or a pharmaceutically acceptable salt, isomer,polymorph, metabolite, hydrate, solvate or ester thereof; etc. which iseffective to treat the disorder. Hyper-proliferative disorders includebut are not limited, e.g., psoriasis, keloids, and other hyperplasiasaffecting the skin, benign prostate hyperplasia (BPH), solid tumors,such as cancers of the breast, respiratory tract, brain, reproductiveorgans, digestive tract, urinary tract, eye, liver, skin, head and neck,thyroid, parathyroid and their distant metastases. Those disorders alsoinclude lymphomas, sarcomas, and leukemias.

Examples of breast cancer include, but are not limited to invasiveductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ,and lobular carcinoma in situ.

Examples of cancers of the respiratory tract include, but are notlimited to small-cell and non-small-cell lung carcinoma, as well asbronchial adenoma and pleuropulmonary blastoma.

Examples of brain cancers include, but are not limited to brain stem andhypophtalmic glioma, cerebellar and cerebral astrocytoma,medulloblastoma, ependymoma, as well as neuroectodermal and pinealtumor.

Tumors of the male reproductive organs include, but are not limited toprostate and testicular cancer. Tumors of the female reproductive organsinclude, but are not limited to endometrial, cervical, ovarian, vaginal,and vulvar cancer, as well as sarcoma of the uterus.

Tumors of the digestive tract include, but are not limited to anal,colon, colorectal, esophageal, gallbladder, gastric, pancreatic, rectal,small-intestine, and salivary gland cancers.

Tumors of the urinary tract include, but are not limited to bladder,penile, kidney, renal pelvis, ureter, urethral and human papillary renalcancers.

Eye cancers include, but are not limited to intraocular melanoma andretinoblastoma.

Examples of liver cancers include, but are not limited to hepatocellularcarcinoma (liver cell carcinomas with or without fibrolamellar variant),cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixedhepatocellular cholangiocarcinoma.

Skin cancers include, but are not limited to squamous cell carcinoma,Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, andnon-melanoma skin cancer.

Head-and-neck cancers include, but are not limited to laryngeal,hypopharyngeal, nasopharyngeal, oropharyngeal cancer, lip and oralcavity cancer and squamous cell. Lymphomas include, but are not limitedto AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-celllymphoma, Burkitt lymphoma, Hodgkin's disease, and lymphoma of thecentral nervous system.

Sarcomas include, but are not limited to sarcoma of the soft tissue,osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, andrhabdomyosarcoma.

Leukemias include, but are not limited to acute myeloid leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia, chronicmyelogenous leukemia, and hairy cell leukemia.

These disorders have been well characterized in humans, but also existwith a similar etiology in other mammals, and can be treated byadministering pharmaceutical compositions of the present invention.

The term “treating” or “treatment” as stated throughout this document isused conventionally, e.g., the management or care of a subject for thepurpose of combating, alleviating, reducing, relieving, improving thecondition of, etc., of a disease or disorder, such as a carcinoma.

Methods of Treating Kinase Disorders

The present invention also provides methods for the treatment ofdisorders associated with aberrant mitogen extracellular kinaseactivity, including, but not limited to stroke, heart failure,hepatomegaly, cardiomegaly, diabetes, Alzheimer's disease, cysticfibrosis, symptoms of xenograft rejections, septic shock or asthma.

Effective amounts of compounds of the present invention can be used totreat such disorders, including those diseases (e.g., cancer) mentionedin the Background section above. Nonetheless, such cancers and otherdiseases can be treated with compounds of the present invention,regardless of the mechanism of action and/or the relationship betweenthe kinase and the disorder.

The phrase “aberrant kinase activity” or “aberrant tyrosine kinaseactivity,” includes any abnormal expression or activity of the geneencoding the kinase or of the polypeptide it encodes. Examples of suchaberrant activity, include, but are not limited to, over-expression ofthe gene or polypeptide; gene amplification; mutations which produceconstitutively-active or hyperactive kinase activity; gene mutations,deletions, substitutions, additions, etc.

The present invention also provides for methods of inhibiting a kinaseactivity, especially of mitogen extracellular kinase, comprisingadministering an effective amount of a compound of the presentinvention, including salts, polymorphs, metabolites, hydrates, solvates,prodrugs (e.g.: esters) thereof, and diastereoisomeric forms thereof.Kinase activity can be inhibited in cells (e.g., in vitro), or in thecells of a mammalian subject, especially a human patient in need oftreatment.

Methods of Treating Angiogenic Disorders

The present invention also provides methods of treating disorders anddiseases associated with excessive and/or abnormal angiogenesis.

Inappropriate and ectopic expression of angiogenesis can be deleteriousto an organism. A number of pathological conditions are associated withthe growth of extraneous blood vessels. These include, e.g., diabeticretinopathy, ischemic retinal-vein occlusion, and retinopathy ofprematurity [Aiello et al. New Engl. J. Med. 1994, 331, 1480; Peer etal. Lab. Invest. 1995, 72, 638], age-related macular degeneration [AMD;see, Lopez et al. Invest. Opththalmol. Vis. Sci. 1996, 37, 855],neovascular glaucoma, psoriasis, retrolental fibroplasias, angiofibroma,inflammation, rheumatoid arthritis (RA), restenosis, in-stentrestenosis, vascular graft restenosis, etc. In addition, the increasedblood supply associated with cancerous and neoplastic tissue, encouragesgrowth, leading to rapid tumor enlargement and metastasis. Moreover, thegrowth of new blood and lymph vessels in a tumor provides an escaperoute for renegade cells, encouraging metastasis and the consequencespread of the cancer. Thus, compounds of the present invention can beutilized to treat and/or prevent any of the aforementioned angiogenesisdisorders, e.g., by inhibiting and/or reducing blood vessel formation;by inhibiting, blocking, reducing, decreasing, etc. endothelial cellproliferation or other types involved in angiogenesis, as well ascausing cell death or apoptosis of such cell types.

Dose and Administration

Based upon standard laboratory techniques known to evaluate compoundsuseful for the treatment of hyper-proliferative disorders and angiogenicdisorders, by standard toxicity tests and by standard pharmacologicalassays for the determination of treatment of the conditions identifiedabove in mammals, and by comparison of these results with the results ofknown medicaments that are used to treat these conditions, the effectivedosage of the compounds of this invention can readily be determined fortreatment of each desired indication. The amount of the activeingredient to be administered in the treatment of one of theseconditions can vary widely according to such considerations as theparticular compound and dosage unit employed, the mode ofadministration, the period of treatment, the age and sex of the patienttreated, and the nature and extent of the condition treated.

The total amount of the active ingredient to be administered willgenerally range from about 0.001 mg/kg to about 200 mg/kg body weightper day, and preferably from about 0.01 mg/kg to about 20 mg/kg bodyweight per day. Clinically useful dosing schedules will range from oneto three times a day dosing to once every four weeks dosing. Inaddition, “drug holidays” in which a patient is not dosed with a drugfor a certain period of time, may be beneficial to the overall balancebetween pharmacological effect and tolerability. A unit dosage maycontain from about 0.5 mg to about 1500 mg of active ingredient, and canbe administered one or more times per day or less than once a day. Theaverage daily dosage for administration by injection, includingintravenous, intramuscular, subcutaneous and parenteral injections, anduse of infusion techniques will preferably be from 0.01 to 200 mg/kg oftotal body weight. The average daily rectal dosage regimen willpreferably be from 0.01 to 200 mg/kg of total body weight. The averagedaily vaginal dosage regimen will preferably be from 0.01 to 200 mg/kgof total body weight. The average daily topical dosage regimen willpreferably be from 0.1 to 200 mg administered between one to four timesdaily. The transdermal concentration will preferably be that required tomaintain a daily dose of from 0.01 to 200 mg/kg. The average dailyinhalation dosage regimen will preferably be from 0.01 to 100 mg/kg oftotal body weight.

Of course the specific initial and continuing dosage regimen for eachpatient will vary according to the nature and severity of the conditionas determined by the attending diagnostician, the activity of thespecific compound employed, the age and general condition of thepatient, time of administration, route of administration, rate ofexcretion of the drug, drug combinations, and the like. The desired modeof treatment and number of doses of a compound of the present inventionor a pharmaceutically acceptable salt or ester or composition thereofcan be ascertained by those skilled in the art using conventionaltreatment tests.

Preferably, the diseases of said method are haematological tumours,solid tumour and/or metastases thereof.

The compounds of the present invention can be used in particular intherapy and prevention, i.e. prophylaxis, of tumour growth andmetastases, especially in solid tumours of all indications and stageswith or without pre-treatment of the tumour growth.

Methods of testing for a particular pharmacological or pharmaceuticalproperty are well known to persons skilled in the art.

The example testing experiments described herein serve to illustrate thepresent invention and the invention is not limited to the examplesgiven.

Biological Evaluation

The utility of the compounds of the present invention can beillustrated, for example, by their activity in vitro in the in vitrotumor cell proliferation assay described below. The link betweenactivity in tumor cell proliferation assays in vitro and anti-tumoractivity in the clinical setting has been very well established in theart. For example, the therapeutic utility of taxol (Silvestrini et al.Stem Cells 1993, 11(6), 528-35), taxotere (Bissery et al. Anti CancerDrugs 1995, 6(3), 339), and topoisomerase inhibitors (Edelman et al.Cancer Chemother. Pharmacol. 1996, 37(5), 385-93) were demonstrated withthe use of in vitro tumor proliferation assays.

Demonstration of the activity of the compounds of the present inventionmay be accomplished through in vitro, ex vivo, and in vivo assays thatare well known in the art. For example, to demonstrate the activity ofthe compounds of the present invention, the following assays may beused.

Biological Assays In Vitro Tumor Cell Proliferation Assays: Cell TiterGlo Proliferation Assay

The adherent tumor cell proliferation assay used to test the compoundsof the present invention involves a readout called Cell Titre-Glodeveloped by Promega (Cunningham, B A “A Growing Issue: CellProliferation Assays. Modern kits ease quantification of cell growth”The Scientist 2001, 15(13), 26, and Crouch, S P et al., “The use of ATPbioluminescence as a measure of cell proliferation and cytotoxicity”Journal of Immunological Methods 1993, 160, 8 1-88).

Assay 1: HCT116 Cell Titer Glo (CTG) Proliferation Assay:

HCT116 cells [human colorectal cell line, expressing mutant BRAF V600E]were plated at a density of 3000 cells/well in 96 well black-clearbottom tissue culture plates (Costar 3603 black/clear bottom) in 100μl/well DMEM medium (DMEM/Ham's F12) with 10% Fetal Bovine Serum (FBS)and stable Glutamine incubated at 37° C. Sister wells were plated inseparate plate for time zero determination. All plates were incubatedovernight at 37° C. Take down time zero plate: 100 μl/well CTG solution(Promega Cell Titer Glo solution) were added to time zero wells insister plate; the plates were mixed for 2 min on orbital shaker toensure cell lysis, incubated for 10 minutes, luminescence was read onVICTOR3 (Perkin Elmer). Twenty-four hours after cell seeding, testcompounds were diluted in 50 μl medium and were added at a finalconcentration range from as high 10 μM to as low 300 μM depending on theactivities of the tested compounds in serial dilutions at a final DMSOconcentration of 0.4%. Cells were incubated for 72 hours at 37° C. afteraddition of the test compound. Then, using a Promega Cell Titer GloLuminescent® assay kit, 100 μl microliter lysis buffer containing of theenzyme luciferase and its substrate, luciferin mixture, were added toeach well and incubated for 10 min at room temperature in the dark tostabilize luminescence signal. The samples were read on VICTOR3 (PerkinElmer) using Luminescence protocol. The percentage change in cell growthwas calculated by normalizing the measurements to the extinctions of thezero point plate (=0%) and the extinction of the untreated (0 μM) cells(=100%). The IC50 values were determined by means of a 4-parameter fitusing the company's own software.

Assay 2: A549 Cell Titer Glo (CTG) Proliferation Assay:

A549 cells [human non small cell lung cancer cell line, expressingmutant K-Ras G12S] were seeded at a density of 2000 cells/well in 96well black-clear bottom tissue culture plates (Costar 3603 black/clearbottom) in 100 μl/well DMEM medium (DMEM/Ham's F12) with 10% FetalBovine Serum (FBS) and stable Glutamine incubated at 37° C. Cell TiterGlo proliferation assays for A549 cells were performed with the sameprotocol as described afore for HCT116 cells.

Assay 3: Colo205 Cell Titer Glo (CTG) Proliferation Assay:

Colo205 cells were plated in RPMI 1640 growth medium supplemented with10% FBS at 3,000 cells per well in 96-well tissue culture plates. Cellswere incubated overnight in a humidified incubator containing 5% CO₂ at37° C. The following day, test compounds were added to wells, seriallydiluted in RPMI 1640 medium containing 10% FBS and 0.03% DMSO and theplates were incubated for 72 h at 37° C. Evaluation of cell density wasmade at different time points (0 and 72 h post-dosing) by adding to eachwell 150 μl of Cell Titer Glo reagent (cat# G7572, Promega, MadisonWis.) followed by incubation of the plates on a rotator for 10 min atroom temperature and then reading of the luminescence on a Victor3instrument. Data analysis was performed using Analyze5 software for IC₅₀analysis.

Assay 4: A375 Cell Titer Glo (CTG) Proliferation Assay:

A375 cells [human malignant melanoma cells, ATCC # CRL-1619, expressingmutant BRAF V600E] were plated at a density of 3000 cells/well in 96well black-clear bottom tissue culture plates (Costar 3603 black/clearbottom) in 100 μL/well DMEM medium (Biochrom; FG0435; +3.7 g/L odiumbicarbonate; +4.5 g/L D-Glucose) with 10% Fetal Bovine Serum (FBS) andstable Glutaminincubated at 37° C. Plate sister wells in separate platefor time zero determination. Incubate all plates overnight 37° C. Takedown time zero plate: add 67 μL/well CTG solution (Promega Cell TiterGlo solution) to time zero wells in sister plate; the plates were mixedfor 2 min on orbital shaker to ensure cell lysis, incubate 10 minutes,read luminescence on VICTOR3 (Perkin Elmer). Twenty-four hours aftercell seeding, test compounds diluted in 50 μL medium are added at afinal concentration range from as high 10 μM to as low 300 μM dependingon the activities of the tested compounds in serial dilutions at a finalDMSO concentration of 0.4%. Cells were incubated for 72 hours at 37° C.after addition of the test compound. Then, using a Promega Cell TiterGlo Luminescent® assay kit, 100 microliters lysis buffer containing ofthe enzyme luciferase and its substrate, luciferin mixture, were addedto each well and incubated for 10 min at room temperature in the dark tostabilize luminescence signal. The samples were read on VICTOR3 (PerkinElmer) using Luminescence protocol. The percentage change in cell growthwas calculated by normalizing the measurements to the extinctions of thezero point plate (=0%) and the extinction of the untreated (0 μM) cells(=100%). The IC50 values were determined by means of a 4-parameter fitusing the company's own software.

Alternatively, the cell proliferation was measured by crystal violet(CV) staining:

Assay 5: A375 Crystal Violet (CV) Proliferation Assay:

Cell proliferation for A375 cells [human melanoma cell line, expressingmutant BRAF V600E] was measured by crystal violet (CV) staining:Cultivated human A375 cells were plated out in a density of 1500cells/measurement point in 200 μl of growth medium (DMEM/HAMS F12 with10% FBS and 2 mM Glutamine) in a 96-well multititer plate. After 24hours, the cells from a plate (zero plate) were stained with crystalviolet (see below), while the medium in the other plates was replaced byfresh culture medium (200 μl) to which the test substances had beenadded in various concentrations (0 μM, and in the range 0.3 nM-30 μM;the final concentration of the solvent dimethyl sulphoxide was 0.5%).The cells were incubated in the presence of the test substances for 4days. The cell proliferation was determined by staining the cells withcrystal violet: the cells were fixed by adding 20 μl/measurement pointof an 11% glutaraldehyde solution at room temperature for 15 min. Afterthe fixed cells had been washed three times with water, the plates weredried at room temperature. The cells were stained by adding 100μl/measurement point of a 0.1% crystal violet solution (pH adjusted topH 3 by adding acetic acid). After the stained cells had been washedthree times with water, the plates were dried at room temperature. Thedye was dissolved by adding 100 μl/measurement point of a 10% aceticacid solution, and the extinction was determined by photometry at awavelength of 595 nm. The percentage change in cell growth wascalculated by normalizing the measurements to the extinctions of thezero point plate (=0%) and the extinction of the untreated (0 μM) cells(=100%). The IC50 values were determined by means of a 4-parameter fitusing the company's own software.

Alternatively, crystal violet (CV) staining assay may be carried out asfollows:

Assay 6: Alternative Conditions for A375 Crystal Violet (CV)Proliferation Assay:

Cultivated human A375 cells were plated out in a density of 1500cells/measurement point in 200 μl of growth medium (DMEM/HAMS F12(Biochrom; FG4815) with 10% FBS and 2 mM Glutamine) in a 96-wellmultititer plate. After 24 hours, the cells from a plate (zero plate)were stained with crystal violet (see below), while the medium in theother plates was replaced by fresh culture medium (200 μl) to which thetest substances had been added in various concentrations (0 μM, and inthe range 0.3 nM-30 μM; the final concentration of the solvent dimethylsulphoxide was 0.5%). The cells were incubated in the presence of thetest substances for 4 days. The cell proliferation was determined bystaining the cells with crystal violet: the cells were fixed by adding20 μl/measurement point of an 11% glutaraldehyde solution at roomtemperature for 15 min. After the fixed cells had been washed threetimes with water, the plates were dried at room temperature. The cellswere stained by adding 100 μl/measurement point of a 0.1% crystal violetsolution (pH adjusted to pH 3 by adding acetic acid). After the stainedcells had been washed three times with water, the plates were dried atroom temperature. The dye was dissolved by adding 100 μl/measurementpoint of a 10% acetic acid solution, and the extinction was determinedby photometry at a wavelength of 595 nm. The percentage change in cellgrowth was calculated by normalizing the measurements to the extinctionsof the zero point plate (=0%) and the extinction of the untreated (0 μM)cells (=100%). The IC50 values were determined by means of a 4-parameterfit using the company's own software.

In vitro inhibition of proliferation of further cancer cell lines can bemeasured in analogy to the afore-described procedures. Details forexemplary further tumor cells lines are given below:

cell Indication Ras or number (all Raf per Cells human) Mutation Methodwell Medium A-431 epidermoid wildtype CTG 3000 DMEM/HAMS F12 cancer(Biochrom; FG4815) + 10% FBS and stable Glutamin A-431 epidermoidwildtype CTG 3000 DMEM/HAMS F12 non- cancer (Biochrom; FG4815) + 10%adherent FBS and stable Glutamin (Plates were coated withpoly-2-hydroxy- ethylmethacrylate before cell seeding) Colo-205 colonBRAF CTG 3000 RPMI1640 (Biochrom; carcinoma V600E FG1215) + 10% heatinactivated FBS and stable glutamin + 1x non- essentiell amino acid + 1mM Sodiumpyruvat + 10 mM Hepes HT-29 colon BRAF CTG 2000 DMEM/HAMS F12cancer V600E (Biochrom; FG4815) + 10% FBS and stable Glutamin Loxmelanoma BRAF CTG 2000 RPMI1640 (Biochrom; V600E FG1215) + 10% heatinactivated FBS and stable glutamin + 1x non- essentiell amino acid + 1mM Sodiumpyruvat MCF-7 breast wildtype CTG 5000 RPMI1640 (F1275; w/ocancer phenol red) + 10% FBS + 2 mM Glutamin + 2 mU/mL Insulin + 1E-10Mestradiol

Further, the following assays may be used to assess the biologicalimportance of the compounds of the present invention:

Assay 6 MEK Biochemical Assay: DELFIA

The DELFIA MEK kinase assay was used to monitor the activity of MEKinhibitors. The kinase reaction was carried out in a 96-wellmicrotitration plate by firstly mixing 70 μL of kinase reaction buffer(50 mM HEPES pH 7.5, 5 mM NaF, 5 mM glycerophosphate, 1 mM sodiumvanadate, 10 mM MgCl₂, 1 mM DTT and 1% (v/v) DMSO) with 20 nM GST-MEK,20 nM His-Raf and 100 nM biotinylated ERK1 (final concentration). Thencompounds with final concentrations of 1 μM, 0.3 μM, 0.1 μM, 0.03 μM,0.01 μM, 0.003 μM, 0.001 μM, 0.0003 μM and 0 μM were added to generatethe dose response inhibition curve. The kinase reaction was started byadding 20 μL of ATP (final concentration 100 μM). After 2 h incubation,the reaction was terminated by adding 20 μl of 0.5 M EDTA. Then 100 μLof the reaction mixture was transferred to a 96 well Streptavidin plate(cat #15120, Pierce Inc. Rockford, Ill.) and subsequently incubated for2 h. After collecting the biotinylated substrate ERK1, the plate waswashed with TBST. An antibody against phospho-p44/42 MAPK (cat#91065,Cell Signaling Technologies, Danvers, Mass.) was added and bond to thephosphorylated substrate. Thereafter, incubation with anEuropium-labeled anti-mouse antibody (cat# AD0124, Wallac Inc, Turku,Finland) followed by a washing step was carried out. The EnhancementSolution was added to dissociate europium ions into solution, where theyformed highly fluorescent chelates with the components of theenhancement solution. The fluorescence of each sample was proportionalto kinase activity and counted on a VICTOR5 instrument (Wallac Inc.).Data analysis was performed using Analyze5 software for IC₅₀ analysis.

Assay 7 MEK1 Activation Kinase Assay

The kinase Cot1 activates MEK1 by phosphorylating its activation loop.The inhibitory activity of compounds of the present invention on thisactivation of MEK1 was quantified employing the HTRF assay described inthe following paragraphs.

N-terminally His6-tagged recombinant kinase domain of the human Cot1(amino acids 30-397, purchased from Millipore, cat. no 14-703) expressedin insect cells (SF21) and purified by Ni-NTA affinity chromatographywas used as kinase. As substrate for the kinase reaction the unactiveC-terminally His6-tagged GST-MEK1 fusion protein (Millipore cat. no14-420) was used.

For the assay 50 nl of a 100 fold concentrated solution of the testcompound in DMSO was pipetted into a black low volume 384 wellmicrotiter plate (Greiner Bio-One, Frickenhausen, Germany), 3 μl of asolution of 24 nM GST-MEK1 and 166.7 μM adenosine-tri-phosphate (ATP) inassay buffer [50 mM Tris/HCl pH 7.5, 10 mM MgCl₂, 2 mM dithiothreitol,0.01% (v/v) Igepal CA 630 (Sigma), 5 mM β-phospho-glycerol] were addedand the mixture was incubated for 10 min at 22° C. to allow pre-bindingof the test compounds to the GST-MEK1 before the start of the kinasereaction. Then the kinase reaction was started by the addition of 2 μlof a solution of Cot1 in assay buffer and the resulting mixture wasincubated for a reaction time of 20 min at 22° C. The concentration ofCot1 in the assay was adjusted depending of the activity of the enzymelot and was chosen appropriate to have the assay in the linear range,typical enzyme concentrations were in the range of about 2 ng/μl (finalconc. in the 5 μl assay volume). The reaction was stopped by theaddition of 5 μl of a solution of HTRF detection reagents (13 nM antiGST-XL665 [#61GSTXLB, Fa. Cis Biointernational, Marcoule, France], 1 nMEu-cryptate labelled anti-phospho-MEK 1/2 (Ser217/221) [#61P17KAZ, Fa.Cis Biointernational],) in an aqueous EDTA-solution (100 mM EDTA, 500 mMKF, 0.2% (w/v) bovine serum albumin in 100 mM HEPES/Sodium hydroxide pH7.5).

The resulting mixture was incubated 2 h at 22° C. to allow the bindingof the phosphorylated GST-MEK1 to the anti-GST-XL665 and the Eu-cryptatelabelled anti-phospho-MEK 1/2 antibody. Subsequently the amount ofSer217/Ser221-phosphorylated substrate was evaluated by measurement ofthe resonance energy transfer from the Eu-Cryptate-labelledanti-phospho-MEK antibody to the anti-GST-XL665. Therefore, thefluorescence emissions at 620 nm and 665 nm after excitation at 350 nmwas measured in a HTRF reader, e.g. a Rubystar (BMG Labtechnologies,Offenburg, Germany) or a Viewlux (Perkin-Elmer). The ratio of theemissions at 665 nm and at 622 nm was taken as the measure for theamount of phosphorylated substrate. The data were normalised (enzymereaction without inhibitor=0% inhibition, all other assay components butno enzyme=100% inhibition). Normally test compound were tested on thesame microtiter plate at 10 different concentrations in the range of 20μM to 1 nM (20 μM, 6.7 μM, 2.2 μM, 0.74 μM, 0.25 μM, 82 nM, 27 nM, 9.2nM, 3.1 nM and 1 nM, dilution series prepared before the assay at thelevel of the 100 fold conc. stock solutions by serial 1:3 dilutions) induplicate values for each concentration and IC₅₀ values were calculatedby a 4 parameter fit using an inhouse software.

Assay 8 Phospho-ERK Mechanistic Assay

A375 and Colo205 cells were plated in RPMI 1640 growth mediumsupplemented with 10% FBS at 25,000 cells per well in 96-well tissueculture plates. Cells were incubated overnight in a humidified incubatorcontaining 5% CO₂ at 37° C. The following day, to prepare the assayplates, anti-rabbit Meso-Scale Discovery (MSD) plates (cat# L41 RA-1,Meso-Scale Discovery, Gaithersburg, Md.) were blocked with 100 μl of 5%MSD blocking buffer for 1 h at room temperature, after which they werewashed three times with 200 μl of TBST buffer. The phospho-ERK rabbitpolyclonal antibody (cat#9101, Cell Signaling Technologies, Danvers,Mass.) diluted at 1:200 into 2.5% of MSD Blocker A-TBST was added (25μl) to each well and the plate was then incubated 1 h at roomtemperature with shaking. The plates were then washed once withphosphate buffered saline (PBS) and ready to receive the cell lysates.While the preparation of the assay plates was ongoing, test compoundswere added to the wells of cell-containing plates from the previous day,serially diluted in RPMI 1640 medium containing 10% FBS, 0.1% bovineserum albumin (BSA) and 0.03% DMSO and the plates were incubated for 1.5h at 37° C. After this incubation, the compound-treated plates werewashed three times with PBS, lysed in 30 μl of Bio-Rad lysis buffer (cat#98601, Bio-Rad Laboratories, Hercules, Calif.) and then left shaking onice for 30 min. The lysates were then loaded on the phospho-ERK coatedMSD plates and the plates Incubated overnight at 4° C. The followingday, the plates were washed three times with TBST and 25 μl of 1:3000diluted total ERK monoclonal antibody (Cat#610123, BD Biosciences, SanDiego, Calif.) was added to the plates that were then incubated 1 h atroom temperature with shaking. After the incubation the plates werewashed three times with TBST as described earlier and 25 μl of MSDsulfo-tag anti-mouse antibody (cat #R32AC-5) diluted 1:1000 were addedinto each well. The plates were Incubated 1 h at room temperature withshaking, then washed four times with TBST. Just prior to reading theplates, 150 μl of MSD Read buffer T was added and the plates were readimmediately on the MSD instrument. Data analysis was performed usingAnalyze5 software for IC₅₀ analysis.

Assay 9

Alternative Conditions for Mechanistic pERK Assay

For the measurement of ERK1/2 phosphorylation in tumor cell lines asingleplex Mesoscale Discovery (MSD) assay is used. This assay is builtup like a sandwich immunoassay. Cell lysates generated from differenttumor cell lines treated with serially diluted MEK inhibitor compoundswere loaded on the MSD plates. Phosphorylated ERK1/2 present in thesamples binds to the capture antibody immobilized on the workingelectrode surface. The sandwich is completed by binding of a detectionantibody to the immobilzed phospho-ERK1/2. This detection antibody islabeled with an electro-chemiluminescent compound. Applying voltage tothe plate electrodes causes the labels, bound to the electrode surfacevia the antibody-phospho ERK1/2 sandwich complex, to emit light. Themeasurement of the emitted light allows a quantitative determination ofthe amount of phosphorylated ERK1/2 present in the sample. In detail, alinear range for the measurement of phosphoERK signals must bedetermined for every cell line used in the assay by titrating differentcell numbers. For the final assay, the previously determined cell numberis seeded in 96 well plates. 24 h after seeding, cells were treated for1.5 h with serially diluted allosteric MEK inhibitor compounds beforethe cells were lysed and lysates were transferred in the MSD assayplate. The manufacturer's protocol was changed in that the binding stepof the phosphorylated ERK to the capture antibody was performed overnight at 4° C. instead of 3 h at room temperature, leading to a bettersignal strength.

A375 or Colo205 cells were plated in 50 μL DMEM growth medium (BiochromFG 0435) supplemented with 10% FBS (Biochrom #S0410) (A375),respectively in RPMI growth medium (Biochrom FG1215) supplemented with10% FBS (Biochrom #S0410), 10 mM HEPES (Biochrom L1613), 4.5 g/L Glucoseand 1 mM sodiumpyruvat (Biochrom L0473) (Colo-205) at 45000 cells perwell in 96-well tissue culture plates. Cells were incubated overnight ina humidified incubator containing 5% CO₂ at 37° C.

The Phospho-ERK by Mesoscale Discovery (MSD) (# K111DWD) assay wasperformed according to the manufacturer's recommendations. In brief theprotocol was:

The day after cell seeding, to prepare the assay plates, MSD wereblocked with 150 μl of MSD blocking buffer for 1 h at room temperature,after which they were washed four times with 150 μl of Tris Wash buffer.While the preparation of the assay plates was ongoing, test compoundswere added to the wells of cell-containing plates from the previous day,serially diluted in respective growth medium containing 10% FBS and 0.1%DMSO and the plates were incubated for 1.5-2 h at 37° C. After thisincubation the medium was aspirated, cells were lysed in 50 μl lysisbuffer and then left shaking for 30 min at 4° C. 25 μL of the lysateswere then loaded on the blocked MSD plates and the plates Incubatedovernight at 4° C. The following day, the plates were washed four timeswith Tris wash buffer and 25 μl detection antibody solution was added tothe plates that were then incubated 1 h at room temperature withshaking. After the incubation the plates were washed four times withTris wash buffer 150 μl of MSD Read buffer T was added and the plateswere read immediately on the MSD instrument. Data analysis was performedusing an in-house software for IC50 analysis.

Assay 10 In Vivo Efficacy Studies: Staged Human Xenograft Models

The in vivo anti-tumor activity of lead compounds was assessed in miceusing xenograft models of human BRAF mutant melanoma and coloncarcinomas. The Female athymic NCR nude mice were implantedsubcutaneously with either a human melanoma (LOX), or a human colon(Colo205) carcinoma lines acquired from American Type Culture Collection(ATCC, Maryland). Treatment was initiated when tumors reachedapproximately 100 mg in size. Compounds were administered orally andfreshly prepared in PEG/water (80%/20% respectively). The general healthof mice was monitored and mortality was recorded daily. Tumor dimensionsand body weights were recorded twice a week starting with the first dayof treatment. Animals were euthanized according to Bayer IACUCguidelines. Treatments producing greater than 20% lethality and/or 20%net body weight loss were considered ‘toxic’.

Tumor growth was measured with electronic calipers three times a weekand tumor weight (mg) calculated according to the following formula:[length (mm)×width (mm)²]/2. Anti-tumor efficacy was determined as afunction of tumor growth inhibition (% TGI). TGI is calculated on daysof measurement using the following formula: (100−mean tumor value oftreated (T)/mean tumor of control value (C)×100)=% T/C. The control usedin the calculations is either the “untreated control” or “vehicle”,whichever provides the most conservative representation of the data. Acompound demonstrating a TGI of greater than or equal to 50% isconsidered active. Statistical significance is determined using either aone-tailed or two-tailed Student's T-Test. The compounds that weretested showed significant dose-dependent tumor growth inhibition in bothLOX and Colo205 models.

Compounds of the invention were tested for activity using one or more ofthe assay procedures presented above.

The following Table shows IC50 values obtained in the above-mentionedAssays 1, 2 and 5, for compounds of the present invention, in comparisonto IC50 values for a compound of prior art document WO 2008/138639, asfollows:

A375 HCT116 A549 Proliferation Proliferation Proliferation Assay (CV)Assay (CTG) Assay (CTG) [Assay 5] [Assay 1] [Assay 2] Example NoStructure IC₅₀ [M] IC₅₀ [M] IC₅₀ [M] Reference example cmpd 6.7 from WO2008138639

4.53E−9  2.08E−7 3.19E−7  1

3.51E−10 2.65E−8 1.81E−8  6

2.44E−9   1.0E−7 6.89E−8  7

1.71E−10  3.0E−9  8

 6.8E−10  1.0E−8 4.78E−9  9

2.39E−9  5.96E−8 1.37E−7  3

1.56E−9  3.17E−8 7.36E−8  4

1.56E−8  5.37E−8 9.25E−8  2

8.99E−10 2.83E−8 3.94E−8 10

7.93E−9   7.0E−8 8.67E−8 11

7.04E−9 6.65E−9 12

7.29E−10  3.2E−8 3.61E−8 14

1.86E−8 

It is believed that one skilled in the art, using the precedinginformation and information available in the art, can utilize thepresent invention to its fullest extent. Those skilled in the art willrecognize that the invention may be practiced with variations on thedisclosed structures, materials, compositions and methods withoutdeparting from the spirit or scope of the invention as it is set forthherein and such variations are regarded as within the ambit of theinvention. The compounds described in the examples are intended to berepresentative of the invention, and it will be understood that thescope of the invention is not limited by the scope of the examples. Thetopic headings set forth above are meant as guidance where certaininformation can be found in the application, but are not intended to bethe only source in the application where information on such topics canbe found. All publications and patents cited above are incorporatedherein by reference.

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1. A compound of general formula (I):

in which: R1 is a hydrogen atom or a fluorine atom; R2 is a halogen atom or a C₂-alkynyl group; R3 is an —NH₂, —NH(C₁-C₆-alkyl), —C₁-C₆-alkyl, or —C₃-C₆-cycloalkyl group; R4 is an —NH₂, —NH(C₁-C₆-alkyl), —C₁-C₆-alkyl, or —C₃-C₆-cycloalkyl group; R5 is a halogen atom, or a —C₁-C₆-alkyl or —O—C₁-C₆-alkyl group; A is —(CH₂)_(n)—, in which n=0, or 1; or a tautomer, stereoisomer, N-oxide, salt, hydrate, solvate, metabolite, or prodrug thereof.
 2. The compound according to claim 1, wherein: R1 is a hydrogen atom or a fluorine atom; R2 is a fluorine atom or a C₂-alkynyl group; R3 is an —NH₂, —NH(C₁-C₆-alkyl), —C₁-C₆-alkyl, or —C₃-C₆-cycloalkyl group; R4 is an —NH₂, —NH(C₁-C₆-alkyl), —C₁-C₆-alkyl, or —C₃-C₆-cycloalkyl group; R5 is a fluorine atom or a methyl group; A is —(CH₂)_(n)—, in which n=0, or 1; or a tautomer, stereoisomer, N-oxide, salt, hydrate, solvate, metabolite, or prodrug thereof.
 3. The compound according to claim 1, wherein: R1 is a hydrogen atom or a fluorine atom; R2 is a fluorine atom or a C₂-alkynyl group; R3 is an —NH₂, —NH(C₁-C₆-alkyl), —C₁-C₆-alkyl, or —C₃-C₆-cycloalkyl group; R4 is an —NH₂, methyl, ethyl, n-propyl, iso-propyl, cyclopropyl or cyclobutyl group; R5 is a fluorine atom or a methyl group; A is —(CH₂)_(n)—, in which n=0, or 1; or a tautomer, stereoisomer, N-oxide, salt, hydrate, solvate, metabolite, or prodrug thereof.
 4. The compound according to claim 1, wherein: R1 is a hydrogen atom or a fluorine atom; R2 is a fluorine atom or a C₂-alkynyl group; R3 is an —NH₂, methyl, ethyl, n-propyl, iso-propyl, cyclopropyl or cyclobutyl group; R4 is an —NH₂, methyl, ethyl, n-propyl, iso-propyl, cyclopropyl or cyclobutyl group; R5 is a fluorine atom or a methyl group; A is —(CH₂)_(n)—, in which n=0, or 1; or a tautomer, stereoisomer, N-oxide, salt, hydrate, solvate, metabolite, or prodrug thereof.
 5. The compound according to claim 1, which is selected from the group consisting of: N-(2-{3-[(ethylsulfonyl)amino]phenoxy}-4-fluoro-6-[(2-fluoro-4-iodophenyl)amino]phenyl)cyclopropanesulfonamide; N-(3-{5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-[(methylsulfonyl)-amino]phenoxy}-phenyl)ethane-sulfonamide; N-(3-{2-[(ethylsulfonyl)amino]-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-phenoxy}phenyl)-ethanesulfonamide; N-(2-{3-[(ethylsulfonyl)amino]phenoxy}-4-fluoro-6-[(2-fluoro-4-iodophenyl)amino]-phenyl)propane-2-sulfonamide; N-(2-{3-[(ethylsulfonyl)amino]phenoxy}-4-fluoro-6-[(2-fluoro-4-iodophenyl)amino]-phenyl)cyclobutane-sulfonamide; N-(3-{5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-(sulfamoylamino)phenoxy}phenyl)ethanesulfonamide; N-(2-{3-[(ethylsulfonyl)amino]-2-methylphenoxy}-4-fluoro-6-[(2-fluoro-4-iodophenyl)amino]phenyl)cyclopropanesulfonamide; N-(3-{5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-(sulfamoylamino)phenoxy}-2-methylphenyl)ethanesulfonamide; N-(2-{3-[(ethylsulfonyl)amino]phenoxy}-6-[(4-ethynyl-2-fluorophenyl)amino]-4-fluorophenyl)cyclopropanesulfonamide; N-(3-[3-[(4-ethynyl-2-fluorophenyl)amino]-5-fluoro-2-(sulfamoylamino)phenoxy]phenyl)ethanesulfonamide [Formic Acid salt]; N-{4-fluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-[3-(sulfamoylamino)phenoxy]phenyl}cyclopropanesulfonamide; N-(4-fluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-{3-[(isopropylsulfonyl)amino]phenoxy}phenyl)cyclopropanesulfonamide; N-(4-fluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-{3-[(methylsulfonyl)amino]phenoxy}phenyl)cyclopropanesulfonamide; N-{4-fluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-[4-fluoro-3-(sulfamoylamino)phenoxy]phenyl}cyclopropanesulfonamide; N-(5-{2-[(cyclopropylsulfonyl)amino]-5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]phenoxy}-2-fluorophenyl)cyclopropanesulfonamide; N-(2-fluoro-5-[5-fluoro-3-[(2-fluoro-4-iodophenyl)amino]-2-(sulfamoylamino)phenoxy]phenyl)sulfuric diamide; and N-(4-fluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-{3-[(sulfamylamino)methyl]phenoxy}phenyl)cyclopropanesulfonamide.
 6. A method of preparing a compound of general formula (I) according to claim 1, said method comprising the step of allowing an intermediate compound of general formula (4):

in which R1, R2, R4, R5 and A are as defined for general formula (I) in claim 1, to react with a sulphonyl chloride of general formula E:

in which R3 is as defined for general formula (I) in claim 1, thereby giving a compound of general formula I:

in which R1, R2, R3, R4, R5 and A are as defined for general formula (I) in claim
 1. 7. A method of preparing a compound of general formula (I) according to claim 1, said method comprising the step of allowing an intermediate compound of general formula (8):

in which R1, R2, R3, R5 and A are as defined for general formula (I) in claim 1, to react with a sulphonyl chloride of general formula D:

in which R4 is as defined for general formula (I) in claim 1, thereby giving a compound of general formula I:

in which R1, R2, R3, R4, R5 and A are as defined for general formula (I) in claim
 1. 8. A method of preparing a compound of general formula (Ic) according to claim 1, said method comprising the step of allowing an intermediate compound of general formula (12):

in which R¹, R², R³, R⁵, and A are as defined for general formula (I) in claim 1, and Pg represents an acid labile protecting group, to react with an acid, thereby giving a compound of formula (Ic):

in which R¹, R², R³, R⁵ and A are as defined for general formula (I) in claim
 1. 9. (canceled)
 10. A pharmaceutical composition comprising a compound of general formula (I), or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same, according to claim 1, and a pharmaceutically acceptable diluent or carrier.
 11. A pharmaceutical combination comprising: one or more compounds of general formula (I), or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same, according to claim 1; and one or more agents selected from: a taxane, such as Docetaxel, Paclitaxel, or Taxol; an epothilone, such as Ixabepilone, Patupilone, or Sagopilone; Mitoxantrone; Predinisolone; Dexamethasone; Estramustin; Vinblastin; Vincristin; Doxorubicin; Adriamycin; Idarubicin; Daunorubicin; Bleomycin; Etoposide; Cyclophosphamide; Ifosfamide; Procarbazine; Melphalan; 5-Fluorouracil; Capecitabine; Fludarabine; Cytarabine; Ara-C; 2-Chloro-2″-deoxyadenosine; Thioguanine; an anti-androgen, such as Flutamide, Cyproterone acetate, or Bicalutamide; Bortezomib; a platinum derivative, such as Cisplatin, or Carboplatin; Chlorambucil; Methotrexate; and Rituximab.
 12. (canceled)
 13. (canceled)
 14. A method for the prophylaxis or treatment of uncontrolled cell growth, proliferation and/or survival, an inappropriate cellular immune response, or an inappropriate cellular inflammatory response, particularly in which the uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune response, or inappropriate cellular inflammatory response is mediated by the mitogen-activated protein kinase (MEK-ERK) pathway, more particularly in which the disease of uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune response, or inappropriate cellular inflammatory response is a haemotological tumour, a solid tumour and/or metastases thereof, e.g. leukaemias and myelodysplastic syndrome, malignant lymphomas, head and neck tumours including brain tumours and brain metastases, tumours of the thorax including non-small cell and small cell lung tumours, gastrointestinal tumours, endocrine tumours, mammary and other gynaecological tumours, urological tumours including renal, bladder and prostate tumours, skin tumours, and sarcomas, and/or metastases thereof comprising administering to a human in need thereof a therapeutically acceptable amount of a compound of general formula (I), or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same, according to claim
 1. 15. A compound of general formula (4):

in which R1, R2, R4, R5 and A are as defined for general formula (I) in claim
 1. 16. A compound of general formula (8):

in which R1, R2, R3, R5 and A are as defined for general formula (I) in claim
 1. 17. A compound of general formula (12):

in which R¹, R², R³, R⁵ and A are as defined for general formula (I) in claim 1 and PG represents an acid-labile protecting group.
 18. (canceled) 